Cyclic depsipeptides and drugs containing the same as the active ingredient

ABSTRACT

Cyclic depsipeptides represented by the formula 
                 
 
wherein:
 
     R is a straight or branched alkyl group of 5-20 carbon atoms or a straight or branched alkoxymethyl group of 5-15-carbon atoms; A, B, D, E and F independently each other are alanine, valine, leucine, isoleucine, phenylalanine, etc.; W and Z independently each other are aspartic acid, asparagine, glutamic acid or glutamine; and m and n independently each other is 0 or 1, or pharmacologically acceptable salts thereof. The present compounds are prepared according to a method conventionally used in peptide synthesis. The present compounds are useful as an agent for promoting the production of apolipoprotein E, a therapeutic agent for neurologic damages, a therapeutic agent for dementia, an agent for inhibiting the production of apolipoprotein B, an agent for promoting the production of apolipoprotein A1 or a therapeutic agent for hyperlipemia.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a cyclic depsipeptide and a pharmaceuticalpreparation containing the same as an active ingredient. The cyclicdepsipeptides of the invention have a promoting activity on theproduction of apolipoprotein E, an inhibitory activity on the productionof apolipoprotein B and a promoting activity on the production ofapolipoprotein A1. Since apolipoprotein E has a repairing activity forneurological damages, the present cyclic depsipeptides having apromoting activity on the production of apolipoprotein E are useful as atherapeutic agent for neurological damages, especially, dementia. On theother hand, apolipoprotein B is a main apolipoprotein of a low-densitylipoprotein cholesterol (LDL cholesterol) known as the “bad” cholesteroland apolipoprotein A1 is a main apolipoprotein of a high-densitylipoprotein cholesterol (HDL cholesterol) known as the “good”cholesterol. Thus, the cyclic depsipeptides of the invention having anactivity of inhibiting the production of apolipoprotein B and anactivity of promoting the production of apolipoprotein A1 are useful asa therapeutic agent for hyperlipemia.

2. Description of the Background

As a therapeutic agent for senile dementia, there have been mainly usedactivators of cerebral circulation and metabolism, but these drugs haveno improving effect on disintegration of the central nervous systemwhich is believed to cause senile dementia. Consequently, they possessno improving effect on dysmnesia and acalculia which are said to becentral symptoms of dementia. In view of the above, there has beendesired a new therapeutic agent for senile dementia which promotes therepair and growth of nervous systems while inhibiting the disintegrationof the central nervous system.

On the other hand, it was reported that apolipoprotein E may begenerated at a high level at the sites of nervous systems which weredamaged and are being repaired (For example, refer to M. J. Igunatius,et al., Proc. Natl. Acad. Sci. U.S.A., 83, 1125 (1986)), which suggeststhat apolipoprotein E will play an important role in repairing nervoussystems.

SUMMARY OF THE INVENTION

We made our earnest studies in order to provide a drug which promotesthe production of apolipoprotein E and has a repairing action onneurological damages. As a result, we have found that a certain cyclicdepsipeptide possesses such actions, upon which the present inventionhas been completed.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to an agent for promoting the productionof apolipoprotein E which contains as an active ingredient a cyclicdepsipeptide represented by the formula (1):

wherein:

R is a straight or branched alkyl group of 5-20 carbon atoms or astraight or branched alkoxymethyl group of 5-15 carbon atoms; A, B, D, Eand F independently each other are a residue of an amino acid selectedfrom alanine, valine, leucine, isoleucine, serine, threonine, lysine,hydroxylysine, arginine, cysteine, methionine, phenylalanine, tyrosine,tryptophan, histidine, proline, 4-hydroxyproline,piperizine-4-carboxylic acid, homoproline, octahydroindole-2-carboxylicacid, norvaline, norleucine, α-t-butylglycine, cyclohexylglycine,azetidine-2-carboxylic acid, 3-(3-pyridyl)alanine,(3-N-methyl)piperizylalanine, 3-(2-naphthyl)alanine,β-cyclohexylalanine, β-t-butylalanine, 9-anthracenylalanine,α-methylalanine and 2-aminobutanoic acid or an N—(C₁-C₄) alkylderivative of said amino acid residue; W and Z independently each otherare a residue of an amino acid selected from aspartic acid, asparagine,glutamic acid and glutamine; and m and n independently each other is 0or 1; and wherein a free amino group, a free carboxyl group or a freeω-carbamido group of said amino acid residue may be protected by aprotecting group commonly used in peptide chemistry and, when said aminoacid residue in the above A, B, D, E, F, W and Z is a residue of lysine,hydroxylysine, glutamic acid or aspartic acid, the amino group orcarboxyl group capable of being bound to an adjacent amino acid bypeptide linkage may be located at either the α-position or theω-position) or a pharmacologically acceptable salt thereof.

The invention also relates to a method for promoting the production ofapolipoprotein E which comprises administering a cyclic depsipeptiderepresented by the above formula (1) or a pharmacologically acceptablesalt thereof.

Further, the invention relates to a therapeutic agent for neurologicdamages or an antidementia agent which comprises as an active ingredienta cyclic depsipeptide represented by the above formula (1) or apharmacologically acceptable salt thereof.

The invention also relates to a method for the treatment of neurologicdamages or dementia which comprises administering a cyclic depsipeptiderepresented by the above formula (1) or a pharmacologically acceptablesalt thereof.

Moreover, the invention relates to a cyclic depsipeptide represented bythe following formula (1′):

(wherein A, B, D, E, F, W, Z, m and n are as defined above, and R′ hasthe same meanings as the above R; provided that there are excluded thecases wherein m and n are 1, A is isoleucine, B is leucine, W isaspartic acid, D is valine, E is leucine, F is leucine, Z is glutamicacid or glutamine, R′ is a group of the formula R₁—(CH₂)_(p)— (whereinR₁ is methyl, isopropyl, sec-butyl or isobutyl and p is an integer of5-15)) or a pharmacologically acceptable salt thereof, and an inhibitoryagent on the production of apolipoprotein B and a promoting agent forthe production of apolipoprotein A1 or an antihyperlipemic agent.

The invention is also concerned with a method for inhibiting theproduction of apolipoprotein B, a method for promoting the production ofapolipoprotein A1 and a method for treating hyperlipemia.

The aforementioned amino acids, of which the cyclic depsipeptides havingthe formula (1) of this invention are composed, may be any of L-isomerand D-isomer.

In the above formula (1), it is preferable that A is Ile or Ala, B isLeu, Ala, t-BuAla, Val or Phe, D is Val or Ala, E is Leu, Ala, t-BuAla,Val or Phe, F is Leu, Ala, t-BuAla, Val or Phe, W is Asp or Glu, Z isGln or Asn, and m and n are 1, and R is a straight alkyl or alkoxymethylgroup of 6-12 carbon atoms. The amino acids for A, D, F, W and Z may bepreferably in the form of L-isomer, while the amino acids for B and Emay be preferably in the form of D-isomer. In particular, it ispreferable that A is Ile or Ala, B is D-Leu, D-Ala, D-t-BuAla, D-Val orD-Phe, D is Val or Ala, E is D-Leu, D-Ala, D-t-BuAla, D-Val or D-Phe, Fis Leu, Ala, t-BuAla, Val or Phe, W is Asp or Glu and Z is Gln or Asn.

Preferable examples of the compounds of the formula (1) may include thecyclic depsipeptides represented by the following formulae orpharmacologically acceptable salts thereof (SEQ ID NOS: 8-19):

In the above formulae, R is as defined above.

As preferable examples of the cyclic depsipeptides represented by theformula (1) or (1′) according to this invention, there may be mentionedthose compounds wherein R is a straight alkyl or alkoxymethyl group of6-12 carbon atoms, m and n are 0 or 1, and wherein

-   -   A=Ile, B=Leu, W=Asp, D=Ala, E=Leu, F=Leu and Z=Gln;    -   A=Ile, B=Ala, W=Asp, D=Val, E=Leu, F=Leu and Z=Gln;    -   A=Ala, B=Leu, W=Asp, D=Val, E=Leu, F=Leu and Z=Gln;    -   A=Ile, B=Leu, W=Asp, D=Val, E=Ala, F=Leu and Z=Gln;    -   A=Ile, B=Leu, W=Asp, D=Val, E=Leu, F=Ala and Z=Gln;    -   or the like.

Of the compounds represented by the formula (1) or (1′), particularlypreferable compounds will be listed hereinafter.

In the above formula, R₂ represents a methyl or isopropyl group, R₃represents a direct bond or a group of —OCH₂—, and q is an integer of2-10.

A B W D E F Z m n Ile D-Phe Glu Val D-Leu Leu Asn 1 1 Ile D-Phe Glu ValD-Leu Leu Gln 1 1 Ile D-Phe Glu Val D-Leu Ala Asn 1 1 Ile D-Phe Glu ValD-Leu Ala Gln 1 1 Ile D-Phe Glu Val D-Ala Leu Asn 1 1 Ile D-Phe Glu ValD-Ala Leu Gln 1 1 Ile D-Phe Glu Val D-Ala Ala Asn 1 1 Ile D-Phe Glu ValD-Ala Ala Gln 1 1 Ile D-Phe Glu Val D-Val Leu Asn 1 1 Ile D-Phe Glu ValD-Val Leu Gln 1 1 Ile D-Phe Glu Val D-Val Ala Asn 1 1 Ile D-Phe Glu ValD-Val Ala Gln 1 1 Ile D-Phe Glu Val D-Phe Leu Asn 1 1 Ile D-Phe Glu ValD-Phe Leu Gln 1 1 Ile D-Phe Glu Val D-Phe Ala Asn 1 1 Ile D-Phe Glu ValD-Phe Ala Gln 1 1 Ile D-Phe Glu Ala D-Leu Leu Asn 1 1 Ile D-Phe Glu AlaD-Leu Leu Gln 1 1 Ile D-Phe Glu Ala D-Leu Ala Asn 1 1 Ile D-Phe Glu AlaD-Leu Ala Gln 1 1 Ile D-Phe Glu Ala D-Ala Leu Asn 1 1 Ile D-Phe Glu AlaD-Ala Leu Gln 1 1 Ile D-Phe Glu Ala D-Ala Ala Asn 1 1 Ile D-Phe Glu AlaD-Ala Ala Gln 1 1 Ile D-Phe Glu Ala D-Val Leu Asn 1 1 Ile D-Phe Glu AlaD-Val Leu Gln 1 1 Ile D-Phe Glu Ala D-Val Ala Asn 1 1 Ile D-Phe Glu AlaD-Val Ala Gln 1 1 Ile D-Phe Glu Ala D-Phe Leu Asn 1 1 Ile D-Phe Glu AlaD-Phe Leu Gln 1 1 Ile D-Phe Glu Ala D-Phe Ala Asn 1 1 Ile D-Phe Glu AlaD-Phe Ala Gln 1 1 Ile D-Ala Asp Val D-Leu Leu Asn 1 1 Ile D-Ala Asp ValD-Leu Leu Gln 1 1 Ile D-Ala Asp Val D-Leu Ala Asn 1 1 Ile D-Ala Asp ValD-Leu Ala Gln 1 1 Ile D-Ala Asp Val D-Ala Leu Asn 1 1 Ile D-Ala Asp ValD-Ala Leu Gln 1 1 Ile D-Ala Asp Val D-Ala Ala Asn 1 1 Ile D-Ala Asp ValD-Ala Ala Gln 1 1 Ile D-Ala Asp Val D-Val Leu Asn 1 1 Ile D-Ala Asp ValD-Val Leu Gln 1 1 Ile D-Ala Asp Val D-Val Ala Asn 1 1 Ile D-Ala Asp ValD-Val Ala Gln 1 1 Ile D-Ala Asp Val D-Phe Leu Asn 1 1 Ile D-Ala Asp ValD-Phe Leu Gln 1 1 Ile D-Ala Asp Val D-Phe Ala Asn 1 1 Ile D-Ala Asp ValD-Phe Ala Gln 1 1 Ile D-Ala Asp Ala D-Leu Leu Asn 1 1 Ile D-Ala Asp AlaD-Leu Leu Gln 1 1 Ile D-Ala Asp Ala D-Leu Ala Asn 1 1 Ile D-Ala Asp AlaD-Leu Ala Gln 1 1 Ile D-Ala Asp Ala D-Ala Leu Asn 1 1 Ile D-Ala Asp AlaD-Ala Leu Gln 1 1 Ile D-Ala Asp Ala D-Ala Ala Asn 1 1 Ile D-Ala Asp AlaD-Ala Ala Gln 1 1 Ile D-Ala Asp Ala D-Val Leu Asn 1 1 Ile D-Ala Asp AlaD-Val Leu Gln 1 1 Ile D-Ala Asp Ala D-Val Ala Asn 1 1 Ile D-Ala Asp AlaD-Val Ala Gln 1 1 Ile D-Ala Asp Ala D-Phe Leu Asn 1 1 Ile D-Ala Asp AlaD-Phe Leu Gln 1 1 Ile D-Ala Asp Ala D-Phe Ala Asn 1 1 Ile D-Ala Asp AlaD-Phe Ala Gln 1 1 Ile D-Ala Glu Val D-Leu Leu Asn 1 1 Ile D-Ala Glu ValD-Leu Leu Gln 1 1 Ile D-Ala Glu Val D-Leu Ala Asn 1 1 Ile D-Ala Glu ValD-Leu Ala Gln 1 1 Ile D-Ala Glu Val D-Ala Leu Asn 1 1 Ile D-Ala Glu ValD-Ala Leu Gln 1 1 Ile D-Ala Glu Val D-Ala Ala Asn 1 1 Ile D-Ala Glu ValD-Ala Ala Gln 1 1 Ile D-Ala Glu Val D-Val Leu Asn 1 1 Ile D-Ala Glu ValD-Val Leu Gln 1 1 Ile D-Ala Glu Val D-Val Ala Asn 1 1 Ile D-Ala Glu ValD-Val Ala Gln 1 1 Ile D-Ala Glu Val D-Phe Leu Asn 1 1 Ile D-Ala Glu ValD-Phe Leu Gln 1 1 Ile D-Ala Glu Val D-Phe Ala Asn 1 1 Ile D-Ala Glu ValD-Phe Ala Gln 1 1 Ile D-Ala Glu Ala D-Leu Leu Asn 1 1 Ile D-Ala Glu AlaD-Leu Leu Gln 1 1 Ile D-Ala Glu Ala D-Leu Ala Asn 1 1 Ile D-Ala Glu AlaD-Leu Ala Gln 1 1 Ile D-Ala Glu Ala D-Ala Leu Asn 1 1 Ile D-Ala Glu AlaD-Ala Leu Gln 1 1 Ile D-Ala Glu Ala D-Ala Ala Asn 1 1 Ile D-Ala Glu AlaD-Ala Ala Gln 1 1 Ile D-Ala Glu Ala D-Val Leu Asn 1 1 Ile D-Ala Glu AlaD-Val Leu Gln 1 1 Ile D-Ala Glu Ala D-Val Ala Asn 1 1 Ile D-Ala Glu AlaD-Val Ala Gln 1 1 Ile D-Ala Glu Ala D-Phe Leu Asn 1 1 Ile D-Ala Glu AlaD-Phe Leu Gln 1 1 Ile D-Ala Glu Ala D-Phe Ala Asn 1 1 Ile D-Ala Glu AlaD-Phe Ala Gln 1 1 Ile D-Val Glu Val D-Leu Leu Asn 1 1 Ile D-Val Glu ValD-Leu Leu Gln 1 1 Ile D-Val Glu Val D-Leu Ala Asn 1 1 Ile D-Val Glu ValD-Leu Ala Gln 1 1 Ile D-Val Glu Val D-Ala Leu Asn 1 1 Ile D-Val Glu ValD-Ala Leu Gln 1 1 Ile D-Val Glu Val D-Ala Ala Asn 1 1 Ile D-Val Glu ValD-Ala Ala Gln 1 1 Ile D-Val Glu Val D-Val Leu Asn 1 1 Ile D-Val Glu ValD-Val Leu Gln 1 1 Ile D-Val Glu Val D-Val Ala Asn 1 1 Ile D-Val Glu ValD-Val Ala Gln 1 1 Ile D-Val Glu Val D-Phe Leu Asn 1 1 Ile D-Val Glu ValD-Phe Leu Gln 1 1 Ile D-Val Glu Val D-Phe Ala Asn 1 1 Ile D-Val Glu ValD-Phe Ala Gln 1 1 Ile D-Val Glu Ala D-Leu Leu Asn 1 1 Ile D-Val Glu AlaD-Leu Leu Gln 1 1 Ile D-Val Glu Ala D-Leu Ala Asn 1 1 Ile D-Val Glu AlaD-Leu Ala Gln 1 1 Ile D-Val Glu Ala D-Ala Leu Asn 1 1 Ile D-Val Glu AlaD-Ala Leu Gln 1 1 Ile D-Val Glu Ala D-Ala Ala Asn 1 1 Ile D-Val Glu AlaD-Ala Ala Gln 1 1 Ile D-Val Glu Ala D-Val Leu Asn 1 1 Ile D-Val Glu AlaD-Val Leu Gln 1 1 Ile D-Val Glu Ala D-Val Ala Asn 1 1 Ile D-Val Glu AlaD-Val Ala Gln 1 1 Ile D-Val Glu Ala D-Phe Leu Asn 1 1 Ile D-Val Glu AlaD-Phe Leu Gln 1 1 Ile D-Val Glu Ala D-Phe Ala Asn 1 1 Ile D-Val Glu AlaD-Phe Ala Gln 1 1 Ile D-Leu Glu Val D-Leu Leu Asn 1 1 Ile D-Leu Glu ValD-Leu Leu Gln 1 1 Ile D-Leu Glu Val D-Leu Ala Asn 1 1 Ile D-Leu Glu ValD-Leu Ala Gln 1 1 Ile D-Leu Glu Val D-Ala Leu Asn 1 1 Ile D-Leu Glu ValD-Ala Leu Gln 1 1 Ile D-Leu Glu Val D-Ala Ala Asn 1 1 Ile D-Leu Glu ValD-Ala Ala Gln 1 1 Ile D-Leu Glu Val D-Val Leu Asn 1 1 Ile D-Leu Glu ValD-Val Leu Gln 1 1 Ile D-Leu Glu Val D-Val Ala Asn 1 1 Ile D-Leu Glu ValD-Val Ala Gln 1 1 Ile D-Leu Glu Val D-Phe Leu Asn 1 1 Ile D-Leu Glu ValD-Phe Leu Gln 1 1 Ile D-Leu Glu Val D-Phe Ala Asn 1 1 Ile D-Leu Glu ValD-Phe Ala Gln 1 1 Ile D-Leu Glu Ala D-Leu Leu Asn 1 1 Ile D-Leu Glu AlaD-Leu Leu Gln 1 1 Ile D-Leu Glu Ala D-Leu Ala Asn 1 1 Ile D-Leu Glu AlaD-Leu Ala Gln 1 1 Ile D-Leu Glu Ala D-Ala Leu Asn 1 1 Ile D-Leu Glu AlaD-Ala Leu Gln 1 1 Ile D-Leu Glu Ala D-Ala Ala Asn 1 1 Ile D-Leu Glu AlaD-Ala Ala Gln 1 1 Ile D-Leu Glu Ala D-Val Leu Asn 1 1 Ile D-Leu Glu AlaD-Val Leu Gln 1 1 Ile D-Leu Glu Ala D-Val Ala Asn 1 1 Ile D-Leu Glu AlaD-Val Ala Gln 1 1 Ile D-Leu Glu Ala D-Phe Leu Asn 1 1 Ile D-Leu Glu AlaD-Phe Leu Gln 1 1 Ile D-Leu Glu Ala D-Phe Ala Asn 1 1 Ile D-Leu Glu AlaD-Phe Ala Gln 1 1 Ile D-Phe Asp Val D-Leu Leu Asn 1 1 Ile D-Phe Asp ValD-Leu Leu Gln 1 1 Ile D-Phe Asp Val D-Leu Ala Asn 1 1 Ile D-Phe Asp ValD-Leu Ala Gln 1 1 Ile D-Phe Asp Val D-Ala Leu Asn 1 1 Ile D-Phe Asp ValD-Ala Leu Gln 1 1 Ile D-Phe Asp Val D-Ala Ala Asn 1 1 Ile D-Phe Asp ValD-Ala Ala Gln 1 1 Ile D-Phe Asp Val D-Val Leu Asn 1 1 Ile D-Phe Asp ValD-Val Leu Gln 1 1 Ile D-Phe Asp Val D-Val Ala Asn 1 1 Ile D-Phe Asp ValD-Val Ala Gln 1 1 Ile D-Phe Asp Val D-Phe Leu Asn 1 1 Ile D-Phe Asp ValD-Phe Leu G1n 1 1 Ile D-Phe Asp Val D-Phe Ala Asn 1 1 Ile D-Phe Asp ValD-Phe Ala Gln 1 1 Ile D-Phe Asp Ala D-Leu Leu Asn 1 1 Ile D-Phe Asp AlaD-Leu Leu Gln 1 1 Ile D-Phe Asp Ala D-Leu Ala Asn 1 1 Ile D-Phe Asp AlaD-Leu Ala Gln 1 1 Ile D-Phe Asp Ala D-Ala Leu Asn 1 1 Ile D-Phe Asp AlaD-Ala Leu Gln 1 1 Ile D-Phe Asp Ala D-Ala Ala Asn 1 1 Ile D-Phe Asp AlaD-Ala Ala Gln 1 1 Ile D-Phe Asp Ala D-Val Leu Asn 1 1 Ile D-Phe Asp AlaD-Val Leu Gln 1 1 Ile D-Phe Asp Ala D-Val Ala Asn 1 1 Ile D-Phe Asp AlaD-Val Ala Gln 1 1 Ile D-Phe Asp Ala D-Phe Leu Asn 1 1 Ile D-Phe Asp AlaD-Phe Leu Gln 1 1 Ile D-Phe Asp Ala D-Phe Ala Asn 1 1 Ile D-Phe Asp AlaD-Phe Ala Gln 1 1 Ile D-Val Asp Val D-Leu Leu Asn 1 1 Ile D-Val Asp ValD-Leu Leu Gln 1 1 Ile D-Val Asp Val D-Leu Ala Asn 1 1 Ile D-Val Asp ValD-Leu Ala Gln 1 1 Ile D-Val Asp Val D-Ala Leu Asn 1 1 Ile D-Val Asp ValD-Ala Leu Gln 1 1 Ile D-Val Asp Val D-Ala Ala Asn 1 1 Ile D-Val Asp ValD-Ala Ala Gln 1 1 Ile D-Val Asp Val D-Val Leu Asn 1 1 Ile D-Val Asp ValD-Val Leu Gln 1 1 Ile D-Val Asp Val D-Val Ala Asn 1 1 Ile D-Val Asp ValD-Val Ala Gln 1 1 Ile D-Val Asp Val D-Phe Leu Asn 1 1 Ile D-Val Asp ValD-Phe Leu Gln 1 1 Ile D-Val Asp Val D-Phe Ala Asn 1 1 Ile D-Val Asp ValD-Phe Ala Gln 1 1 Ile D-Val Asp Ala D-Leu Leu Asn 1 1 Ile D-Val Asp AlaD-Leu Leu Gln 1 1 Ile D-Val Asp Ala D-Leu Ala Asn 1 1 Ile D-Val Asp AlaD-Leu Ala Gln 1 1 Ile D-Val Asp Ala D-Ala Leu Asn 1 1 Ile D-Val Asp AlaD-Ala Leu Gln 1 1 Ile D-Val Asp Ala D-Ala Ala Asn 1 1 Ile D-Val Asp AlaD-Ala Ala Gln 1 1 Ile D-Val Asp Ala D-Val Leu Asn 1 1 Ile D-Val Asp AlaD-Val Leu Gln 1 1 Ile D-Val Asp Ala D-Val Ala Asn 1 1 Ile D-Val Asp AlaD-Val Ala Gln 1 1 Ile D-Val Asp Ala D-Phe Leu Asn 1 1 Ile D-Val Asp AlaD-Phe Leu Gln 1 1 Ile D-Val Asp Ala D-Phe Ala Asn 1 1 Ile D-Val Asp AlaD-Phe Ala Gln 1 1 Ile D-Leu Asp Val D-Leu Leu Asn 1 1 Ile D-Leu Asp ValD-Leu Leu Gln 1 1 Ile D-Leu Asp Val D-Leu Ala Asn 1 1 Ile D-Leu Asp ValD-Leu Ala Gln 1 1 Ile D-Leu Asp Val D-Ala Leu Asn 1 1 Ile D-Leu Asp ValD-Ala Leu Gln 1 1 Ile D-Leu Asp Val D-Ala Ala Asn 1 1 Ile D-Leu Asp ValD-Ala Ala Gln 1 1 Ile D-Leu Asp Val D-Val Leu Asn 1 1 Ile D-Leu Asp ValD-Val Leu Gln 1 1 Ile D-Leu Asp Val D-Val Ala Asn 1 1 Ile D-Leu Asp ValD-Val Ala Gln 1 1 Ile D-Leu Asp Val D-Phe Leu Asn 1 1 Ile D-Leu Asp ValD-Phe Leu Gln 1 1 Ile D-Leu Asp Val D-Phe Ala Asn 1 1 Ile D-Leu Asp ValD-Phe Ala Gln 1 1 Ile D-Leu Asp Ala D-Leu Leu Asn 1 1 Ile D-Leu Asp AlaD-Leu Leu Gln 1 1 Ile D-Leu Asp Ala D-Leu Ala Asn 1 1 Ile D-Leu Asp AlaD-Leu Ala Gln 1 1 Ile D-Leu Asp Ala D-Ala Leu Asn 1 1 Ile D-Leu Asp AlaD-Ala Leu Gln 1 1 Ile D-Leu Asp Ala D-Ala Ala Asn 1 1 Ile D-Leu Asp AlaD-Ala Ala Gln 1 1 Ile D-Leu Asp Ala D-Val Leu Asn 1 1 Ile D-Leu Asp AlaD-Val Leu Gln 1 1 Ile D-Leu Asp Ala D-Val Ala Asn 1 1 Ile D-Leu Asp AlaD-Val Ala Gln 1 1 Ile D-Leu Asp Ala D-Phe Leu Asn 1 1 Ile D-Leu Asp AlaD-Phe Leu Gln 1 1 Ile D-Leu Asp Ala D-Phe Ala Asn 1 1 Ile D-Leu Asp AlaD-Phe Ala Gln 1 1 Ala D-Val Glu Val D-Leu Leu Asn 1 1 Ala D-Val Glu ValD-Leu Leu Gln 1 1 Ala D-Val Glu Val D-Leu Ala Asn 1 1 Ala D-Val Glu ValD-Leu Ala Gln 1 1 Ala D-Val Glu Val D-Ala Leu Asn 1 1 Ala D-Val Glu ValD-Ala Leu Gln 1 1 Ala D-Val Glu Val D-Ala Ala Asn 1 1 Ala D-Val Glu ValD-Ala Ala Gln 1 1 Ala D-Val Glu Val D-Val Leu Asn 1 1 Ala D-Val Glu ValD-Val Leu Gln 1 1 Ala D-Val Glu Val D-Val Ala Asn 1 1 Ala D-Val Glu ValD-Val Ala Gln 1 1 Ala D-Val Glu Val D-Phe Leu Asn 1 1 Ala D-Val Glu ValD-Phe Leu Gln 1 1 Ala D-Val Glu Val D-Phe Ala Asn 1 1 Ala D-Val Glu ValD-Phe Ala Gln 1 1 Ala D-Val Glu Ala D-Leu Leu Asn 1 1 Ala D-Val Glu AlaD-Leu Leu Gln 1 1 Ala D-Val Glu Ala D-Leu Ala Asn 1 1 Ala D-Val Glu AlaD-Leu Ala Gln 1 1 Ala D-Val Glu Ala D-Ala Leu Asn 1 1 Ala D-Val Glu AlaD-Ala Leu Gln 1 1 Ala D-Val Glu Ala D-Ala Ala Asn 1 1 Ala D-Val Glu AlaD-Ala Ala Gln 1 1 Ala D-Val Glu Ala D-Val Leu Asn 1 1 Ala D-Val Glu AlaD-Val Leu Gln 1 1 Ala D-Val Glu Ala D-Val Ala Asn 1 1 Ala D-Val Glu AlaD-Val Ala Gln 1 1 Ala D-Val Glu Ala D-Phe Leu Asn 1 1 Ala D-Val Glu AlaD-Phe Leu Gln 1 1 Ala D-Val Glu Ala D-Phe Ala Asn 1 1 Ala D-Val Glu AlaD-Phe Ala Gln 1 1 Ala D-Leu Glu Val D-Leu Leu Asn 1 1 Ala D-Leu Glu ValD-Leu Leu Gln 1 1 Ala D-Leu Glu Val D-Leu Ala Asn 1 1 Ala D-Leu Glu ValD-Leu Ala Gln 1 1 Ala D-Leu Glu Val D-Ala Leu Asn 1 1 Ala D-Leu Glu ValD-Ala Leu Gln 1 1 Ala D-Leu Glu Val D-Ala Ala Asn 1 1 Ala D-Leu Glu ValD-Ala Ala Gln 1 1 Ala D-Leu Glu Val D-Val Leu Asn 1 1 Ala D-Leu Glu ValD-Val Leu Gln 1 1 Ala D-Leu Glu Val D-Val Ala Asn 1 1 Ala D-Leu Glu ValD-Val Ala Gln 1 1 Ala D-Leu Glu Val D-Phe Leu Asn 1 1 Ala D-Leu Glu ValD-Phe Leu Gln 1 1 Ala D-Leu Glu Val D-Phe Ala Asn 1 1 Ala D-Leu Glu ValD-Phe Ala Gln 1 1 Ala D-Leu Glu Ala D-Leu Leu Asn 1 1 Ala D-Leu Glu AlaD-Leu Leu Gln 1 1 Ala D-Leu Glu Ala D-Leu Ala Asn 1 1 Ala D-Leu Glu AlaD-Leu Ala Gln 1 1 Ala D-Leu Glu Ala D-Ala Leu Asn 1 1 Ala D-Leu Glu AlaD-Ala Leu Gln 1 1 Ala D-Leu Glu Ala D-Ala Ala Asn 1 1 Ala D-Leu Glu AlaD-Ala Ala Gln 1 1 Ala D-Leu Glu Ala D-Val Leu Asn 1 1 Ala D-Leu Glu AlaD-Val Leu Gln 1 1 Ala D-Leu Glu Ala D-Val Ala Asn 1 1 Ala D-Leu Glu AlaD-Val Ala Gln 1 1 Ala D-Leu Glu Ala D-Phe Leu Asn 1 1 Ala D-Leu Glu AlaD-Phe Leu Gln 1 1 Ala D-Leu Glu Ala D-Phe Ala Asn 1 1 Ala D-Leu Glu AlaD-Phe Ala Gln 1 1 Ala D-Ala Glu Val D-Leu Leu Asn 1 1 Ala D-Ala Glu ValD-Leu Leu Gln 1 1 Ala D-Ala Glu Val D-Leu Ala Asn 1 1 Ala D-Ala Glu ValD-Leu Ala Gln 1 1 Ala D-Ala Glu Val D-Ala Leu Asn 1 1 Ala D-Ala Glu ValD-Ala Leu Gln 1 1 Ala D-Ala Glu Val D-Ala Ala Asn 1 1 Ala D-Ala Glu ValD-Ala Ala Gln 1 1 Ala D-Ala Glu Val D-Val Leu Asn 1 1 Ala D-Ala Glu ValD-Val Leu Gln 1 1 Ala D-Ala Glu Val D-Val Ala Asn 1 1 Ala D-Ala Glu ValD-Val Ala Gln 1 1 Ala D-Ala Glu Val D-Phe Leu Asn 1 1 Ala D-Ala Glu ValD-Phe Leu Gln 1 1 Ala D-Ala Glu Val D-Phe Ala Asn 1 1 Ala D-Ala Glu ValD-Phe Ala Gln 1 1 Ala D-Ala Glu Ala D-Leu Leu Asn 1 1 Ala D-Ala Glu AlaD-Leu Leu Gln 1 1 Ala D-Ala Glu Ala D-Leu Ala Asn 1 1 Ala D-Ala Glu AlaD-Leu Ala Gln 1 1 Ala D-Ala Glu Ala D-Ala Leu Asn 1 1 Ala D-Ala Glu AlaD-Ala Leu Gln 1 1 Ala D-Ala Glu Ala D-Ala Ala Asn 1 1 Ala D-Ala Glu AlaD-Ala Ala Gln 1 1 Ala D-Ala Glu Ala D-Val Leu Asn 1 1 Ala D-Ala Glu AlaD-Val Leu Gln 1 1 Ala D-Ala Glu Ala D-Val Ala Asn 1 1 Ala D-Ala Glu AlaD-Val Ala Gln 1 1 Ala D-Ala Glu Ala D-Phe Leu Asn 1 1 Ala D-Ala Glu AlaD-Phe Leu Gln 1 1 Ala D-Ala Glu Ala D-Phe Ala Asn 1 1 Ala D-Ala Glu AlaD-Phe Ala Gln 1 1 Ala D-Phe Glu Val D-Leu Leu Asn 1 1 Ala D-Phe Glu ValD-Leu Leu Gln 1 1 Ala D-Phe Glu Val D-Leu Ala Asn 1 1 Ala D-Phe Glu ValD-Leu Ala Gln 1 1 Ala D-Phe Glu Val D-Ala Leu Asn 1 1 Ala D-Phe Glu ValD-Ala Leu Gln 1 1 Ala D-Phe Glu Val D-Ala Ala Asn 1 1 Ala D-Phe Glu ValD-Ala Ala Gln 1 1 Ala D-Phe Glu Val D-Val Leu Asn 1 1 Ala D-Phe Glu ValD-Val Leu Gln 1 1 Ala D-Phe Glu Val D-Val Ala Asn 1 1 Ala D-Phe Glu ValD-Val Ala Gln 1 1 Ala D-Phe Glu Val D-Phe Leu Asn 1 1 Ala D-Phe Glu ValD-Phe Leu Gln 1 1 Ala D-Phe Glu Val D-Phe Ala Asn 1 1 Ala D-Phe Glu ValD-Phe Ala Gln 1 1 Ala D-Phe Glu Ala D-Leu Leu Asn 1 1 Ala D-Phe Glu AlaD-Leu Leu Gln 1 1 Ala D-Phe Glu Ala D-Leu Ala Asn 1 1 Ala D-Phe Glu AlaD-Leu Ala Gln 1 1 Ala D-Phe Glu Ala D-Ala Leu Asn 1 1 Ala D-Phe Glu AlaD-Ala Leu Gln 1 1 Ala D-Phe Glu Ala D-Ala Ala Asn 1 1 Ala D-Phe Glu AlaD-Ala Ala Gln 1 1 Ala D-Phe Glu Ala D-Val Leu Asn 1 1 Ala D-Phe Glu AlaD-Val Leu Gln 1 1 Ala D-Phe Glu Ala D-Val Ala Asn 1 1 Ala D-Phe Glu AlaD-Val Ala Gln 1 1 Ala D-Phe Glu Ala D-Phe Leu Asn 1 1 Ala D-Phe Glu AlaD-Phe Leu Gln 1 1 Ala D-Phe Glu Ala D-Phe Ala Asn 1 1 Ala D-Phe Glu AlaD-Phe Ala Gln 1 1 Ala D-Leu Asp Val D-Leu Leu Asn 1 1 Ala D-Leu Asp ValD-Leu Leu Gln 1 1 Ala D-Leu Asp Val D-Leu Ala Asn 1 1 Ala D-Leu Asp ValD-Leu Ala Gln 1 1 Ala D-Leu Asp Val D-Ala Leu Asn 1 1 Ala D-Leu Asp ValD-Ala Leu Gln 1 1 Ala D-Leu Asp Val D-Ala Ala Asn 1 1 Ala D-Leu Asp ValD-Ala Ala Gln 1 1 Ala D-Leu Asp Val D-Val Leu Asn 1 1 Ala D-Leu Asp ValD-Val Leu Gln 1 1 Ala D-Leu Asp Val D-Val Ala Asn 1 1 Ala D-Leu Asp ValD-Val Ala Gln 1 1 Ala D-Leu Asp Val D-Phe Leu Asn 1 1 Ala D-Leu Asp ValD-Phe Leu Gln 1 1 Ala D-Leu Asp Val D-Phe Ala Asn 1 1 Ala D-Leu Asp ValD-Phe Ala Gln 1 1 Ala D-Leu Asp Ala D-Leu Leu Asn 1 1 Ala D-Leu Asp AlaD-Leu Leu Gln 1 1 Ala D-Leu Asp Ala D-Leu Ala Asn 1 1 Ala D-Leu Asp AlaD-Leu Ala Gln 1 1 Ala D-Leu Asp Ala D-Ala Leu Asn 1 1 Ala D-Leu Asp AlaD-Ala Leu Gln 1 1 Ala D-Leu Asp Ala D-Ala Ala Asn 1 1 Ala D-Leu Asp AlaD-Ala Ala Gln 1 1 Ala D-Leu Asp Ala D-Val Leu Asn 1 1 Ala D-Leu Asp AlaD-Val Leu Gln 1 1 Ala D-Leu Asp Ala D-Val Ala Asn 1 1 Ala D-Leu Asp AlaD-Val Ala Gln 1 1 Ala D-Leu Asp Ala D-Phe Leu Asn 1 1 Ala D-Leu Asp AlaD-Phe Leu Gln 1 1 Ala D-Leu Asp Ala D-Phe Ala Asn 1 1 Ala D-Leu Asp AlaD-Phe Ala Gln 1 1 Ala D-Ala Asp Val D-Leu Leu Asn 1 1 Ala D-Ala Asp ValD-Leu Leu Gln 1 1 Ala D-Ala Asp Val D-Leu Ala Asn 1 1 Ala D-Ala Asp ValD-Leu Ala Gln 1 1 Ala D-Ala Asp Val D-Ala Leu Asn 1 1 Ala D-Ala Asp ValD-Ala Leu Gln 1 1 Ala D-Ala Asp Val D-Ala Ala Asn 1 1 Ala D-Ala Asp ValD-Ala Ala Gln 1 1 Ala D-Ala Asp Val D-Val Leu Asn 1 1 Ala D-Ala Asp ValD-Val Leu Gln 1 1 Ala D-Ala Asp Val D-Val Ala Asn 1 1 Ala D-Ala Asp ValD-Val Ala Gln 1 1 Ala D-Ala Asp Val D-Phe Leu Asn 1 1 Ala D-Ala Asp ValD-Phe Leu Gln 1 1 Ala D-Ala Asp Val D-Phe Ala Asn 1 1 Ala D-Ala Asp ValD-Phe Ala Gln 1 1 Ala D-Ala Asp Ala D-Leu Leu Asn 1 1 Ala D-Ala Asp AlaD-Leu Leu Gln 1 1 Ala D-Ala Asp Ala D-Leu Ala Asn 1 1 Ala D-Ala Asp AlaD-Leu Ala Gln 1 1 Ala D-Ala Asp Ala D-Ala Leu Asn 1 1 Ala D-Ala Asp AlaD-Ala Leu Gln 1 1 Ala D-Ala Asp Ala D-Ala Ala Asn 1 1 Ala D-Ala Asp AlaD-Ala Ala Gln 1 1 Ala D-Ala Asp Ala D-Val Leu Asn 1 1 Ala D-Ala Asp AlaD-Val Leu Gln 1 1 Ala D-Ala Asp Ala D-Val Ala Asn 1 1 Ala D-Ala Asp AlaD-Val Ala Gln 1 1 Ala D-Ala Asp Ala D-Phe Leu Asn 1 1 Ala D-Ala Asp AlaD-Phe Leu Gln 1 1 Ala D-Ala Asp Ala D-Phe Ala Asn 1 1 Ala D-Ala Asp AlaD-Phe Ala Gln 1 1 Ala D-Phe Asp Val D-Leu Leu Asn 1 1 Ala D-Phe Asp ValD-Leu Leu Gln 1 1 Ala D-Phe Asp Val D-Leu Ala Asn 1 1 Ala D-Phe Asp ValD-Leu Ala Gln 1 1 Ala D-Phe Asp Val D-Ala Leu Asn 1 1 Ala D-Phe Asp ValD-Ala Leu Gln 1 1 Ala D-Phe Asp Val D-Ala Ala Asn 1 1 Ala D-Phe Asp ValD-Ala Ala Gln 1 1 Ala D-Phe Asp Val D-Val Leu Asn 1 1 Ala D-Phe Asp ValD-Val Leu Gln 1 1 Ala D-Phe Asp Val D-Val Ala Asn 1 1 Ala D-Phe Asp ValD-Val Ala Gln 1 1 Ala D-Phe Asp Val D-Phe Leu Asn 1 1 Ala D-Phe Asp ValD-Phe Leu Gln 1 1 Ala D-Phe Asp Val D-Phe Ala Asn 1 1 Ala D-Phe Asp ValD-Phe Ala Gln 1 1 Ala D-Phe Asp Ala D-Leu Leu Asn 1 1 Ala D-Phe Asp AlaD-Leu Leu Gln 1 1 Ala D-Phe Asp Ala D-Leu Ala Asn 1 1 Ala D-Phe Asp AlaD-Leu Ala Gln 1 1 Ala D-Phe Asp Ala D-Ala Leu Asn 1 1 Ala D-Phe Asp AlaD-Ala Leu Gln 1 1 Ala D-Phe Asp Ala D-Ala Ala Asn 1 1 Ala D-Phe Asp AlaD-Ala Ala Gln 1 1 Ala D-Phe Asp Ala D-Val Leu Asn 1 1 Ala D-Phe Asp AlaD-Val Leu Gln 1 1 Ala D-Phe Asp Ala D-Val Ala Asn 1 1 Ala D-Phe Asp AlaD-Val Ala Gln 1 1 Ala D-Phe Asp Ala D-Phe Leu Asn 1 1 Ala D-Phe Asp AlaD-Phe Leu Gln 1 1 Ala D-Phe Asp Ala D-Phe Ala Asn 1 1 Ala D-Phe Asp AlaD-Phe Ala Gln 1 1 Ala D-Val Asp Val D-Leu Leu Asn 1 1 Ala D-Val Asp ValD-Leu Leu Gln 1 1 Ala D-Val Asp Val D-Leu Ala Asn 1 1 Ala D-Val Asp ValD-Leu Ala Gln 1 1 Ala D-Val Asp Val D-Ala Leu Asn 1 1 Ala D-Val Asp ValD-Ala Leu Gln 1 1 Ala D-Val Asp Val D-Ala Ala Asn 1 1 Ala D-Val Asp ValD-Ala Ala Gln 1 1 Ala D-Val Asp Val D-Val Leu Asn 1 1 Ala D-Val Asp ValD-Val Leu Gln 1 1 Ala D-Val Asp Val D-Val Ala Asn 1 1 Ala D-Val Asp ValD-Val Ala Gln 1 1 Ala D-Val Asp Val D-Phe Leu Asn 1 1 Ala D-Val Asp ValD-Phe Leu Gln 1 1 Ala D-Val Asp Val D-Phe Ala Asn 1 1 Ala D-Val Asp ValD-Phe Ala Gln 1 1 Ala D-Val Asp Ala D-Leu Leu Asn 1 1 Ala D-Val Asp AlaD-Leu Leu Gln 1 1 Ala D-Val Asp Ala D-Leu Ala Asn 1 1 Ala D-Val Asp AlaD-Leu Ala Gln 1 1 Ala D-Val Asp Ala D-Ala Leu Asn 1 1 Ala D-Val Asp AlaD-Ala Leu Gln 1 1 Ala D-Val Asp Ala D-Ala Ala Asn 1 1 Ala D-Val Asp AlaD-Ala Ala Gln 1 1 Ala D-Val Asp Ala D-Val Leu Asn 1 1 Ala D-Val Asp AlaD-Val Leu Gln 1 1 Ala D-Val Asp Ala D-Val Ala Asn 1 1 Ala D-Val Asp AlaD-Val Ala Gln 1 1 Ala D-Val Asp Ala D-Phe Leu Asn 1 1 Ala D-Val Asp AlaD-Phe Leu Gln 1 1 Ala D-Val Asp Ala D-Phe Ala Asn 1 1 Ala D-Val Asp AlaD-Phe Ala Gln 1 1 Ile D-Leu Asp — — Val Gln 0 0 Ile D-Leu Asp — ValD-Leu Gln 0 1 Ala D-Leu Asp — — Val Gln 0 0 Ala D-Leu Asp — Val D-LeuGln 0 1

In the description of the above formulae and the following Examples andothers, the amino acid or amino acid residue constituting the peptidesis referred to herein by means of the triliteral expression systemgenerally employed for the indication of amino acids.

More specifically, there are applied the following expressions such asalanine=Ala, valine=Val, leucine=Leu, isoleucine=Ile, serine=Ser,threonine=Thr, aspartic acid=Asp, asparagine=Asn, glutamic acid=Glu,glutamine=Gln, lysine=Lys, cysteine=Cys, phenylalanine=Phe,tyrosine=Tyr, tryptophan=Trp, histidine=His, proline=Pro,4-hydroxyproline=4Hyp, etc.

Agr. Biol. Chem., Vol. 33, No. 10, pp. 1523-1524, 1969, discloses thecompound of the above formula (1) wherein m and n are 1, A isisoleucine, B is leucine, W is aspartic acid, D is valine, E is leucine,F is leucine, Z is glutamic acid and R is a group of the formulaR₁—(CH₂)_(p)— (wherein R₁ is isopropyl and p is 9) as a surfactanthaving an anticoagulant activity. Tetrahedron Letters, Vol. 35, No. 31,pp. 5571-5574, 1994, discloses the compound of the above formula (1)wherein m, n, A, B, W, D, E, and F are as defined above, Z is glutamineand R is a group having the formula R₁—(CH₂)_(p)— (wherein R₁ is methyland p is 11) as having an antifungal, antibacterial or anti-tumoractivity. WO 95/32990 discloses the compound of the above formula (1)wherein m, n, A, B, W, D, E, and F are as defined above, Z is glutamineand R is a group having the formula R₁—(CH₂)_(p)— (wherein R₁ isisopropyl and p is 5-15) as a useful antihyperlipemic agent, an agentfor inhibiting lipid secretion, or an agent for inhibiting theproduction of apolipoprotein B. Further, JP-A-7-291993 discloses thatthe compound of the above formula (1) wherein m, n, A, B, W, D, E, and Fare as defined above, Z is glutamine and R is a group of the formulaR₁—(CH₂)_(p)— (wherein R₁ is isopropyl, sec-butyl or isobutyl and p is8) has an endothelin-antagonistic activity and is useful as avasodilating agent, an antihyperlipemic agent and others. However, noneof the above references discloses that these compounds have a promotingactivity on the production of apolipoprotein E.

Of the cyclic depsipeptides of the present invention, the knowncompounds as described above can be produced by cultivating amicroorganism capable of producing said depsipeptides which belongs tothe genus of Bacillus (for example, Bacillus sp. No. 4691 strain (FERMBP-5101) and others) according to the processes as disclosed inW095/32990, JP-A-7-291993 and others.

Alternatively, the cyclic depsipeptides of the invention can be alsoprepared, for example, according to the conventional procedures for thesynthesis of peptides as mentioned below.

More specifically, the cyclic depsipeptides represented by the aboveformula (1) can be prepared according to the following steps comprising:

-   -   condensing a 3-hydroxy-middle chain or -long chain aliphatic        acid having a protected carboxyl group represented by the        formula (3)        (wherein X is a protecting group for carboxyl group and R is as        defined above), which is obtained by protecting the carboxyl        group of a 3-hydroxy-middle chain or -long chain aliphatic acid        represented by the formula (2)        (wherein R is as defined above) with a suitable protecting        group, with an amino acid A having a protected amino group to        form a compound represented by the formula (4)        (wherein Y is an amino-protecting group and A, X and R are as        defined above);

removing the amino-protecting group in the amino acid A of the compoundto form a compound represented by the formula (5)

(wherein A, X and R are as defined above);

condensing the compound with an amino acid B having a protected aminogroup to form a depsipeptide represented by the formula (6)

(wherein A, B, Y, X and R are as defined above);

removing the amino-protecting group in the amino acid B of thedepsipeptide to form a depsipeptide represented by the formula (7)

(wherein A, B, X and R are as defined above);

condensing the depsipeptide with aspartic acid having a protected aminogroup and a protected carboxyl group at the β-position, asparaginehaving a protected amino group and a protected carbamido group at theβ-position, glutamic acid having a protected amino group and a protectedcarboxyl group at the γ-position or glutamine having a protected aminogroup and a protected carbamide group at the γ-position to form adepsipeptide represented by the formula

(wherein X′ is a protecting group for a carboxyl group at the β-positionof aspartic acid, a carboxyl group at the γ-position of glutamic acid, acarbamido group at the β-position of asparagine or a carbamido group atthe γ-position of glutamine and A, B, W, X, Y and R are as definedabove);

removing the amino-protecting group in aspartic acid, asparagine,glutamic acid or glutamine of the depsipeptide to form a depsipeptiderepresented by the formula (9)

(wherein A, B, W, X, X′ and R are as defined above);

condensing the depsipeptide with an amino acid D having a protectedamino group to form a depsipeptide represented by the formula (10)

(wherein A, B, W, D, Y, X, X′ and R are as defined above);

removing the amino-protecting group in the amino acid D of thedepsipeptide to form a depsipeptide represented by the formula (11)

(wherein A, B, W, D, X, X′ and R are as defined above);

condensing the depsipeptide with an amino acid E having a protectedamino group or condensing the depsipeptide of the formula (9) with anamino acid E having a protected amino group, without the condensationstep of the amino acid D, to form a compound represented by the formula(12)

(wherein A, B, W, D, E, Y, X, X′, R and m are as defined above);

removing the amino-protecting group in the amino acid E of thedepsipeptide thus obtained to form a depsipeptide represented by theformula (13)

(wherein A, B, W, D, E, X, X′ and R are as defined above);

condensing the depsipeptide with an amino acid F having a protectedamino group or condensing the depsipeptide of the formula (11) or theformula (9) with an amino acid F having a protected amino group, withoutthe condensation step of the amino acid E, to form a compoundrepresented by the formula (14)

(wherein A, B, W, D, E, F, Y, X, X′, R, m and n are as defined above);

removing the amino-protecting group in the amino acid F of thedepsipeptide thus obtained to form a depsipeptide represented by theformula (15)

(wherein A, B, W, D, E, F, Y, X, X′, R, m and n are as defined above);

condensing the depsipeptide with aspartic acid having a protected aminogroup and a protected carboxyl group at the β-position, asparaginehaving a protected amino group and a protected carbamido group at theβ-position, glutamic acid having a protected amino group and a protectedcarboxyl group at the γ-position or glutamine having a protected aminogroup and a protected carbamide group at the γ-position to form adepsipeptide represented by the formula (16)

(wherein X″ is a protecting group for a carboxyl group at the β-positionof aspartic acid, a carboxyl group at the γ-position of glutamic acid, acarbamido group at the β-position of asparagine or a carbamido group atthe γ-position of glutamine and A, B, W, D, E, F, Z, X, X′, Y, R, m andn are as defined above);

removing the protecting group for the amino group of aspartic acid,asparagine, glutamic acid or glutamine of the depsipeptide to form adepsipeptide represented by the formula (17)

(wherein A, B, W, D, E, F, Z, X, X′, X″, R, m and n are as definedabove);

removing the carboxyl-protecting group X of the depsipeptide followed byself-condensation to form a cyclic depsipeptide represented by theformula (18)

(wherein A, B, W, D, E, F, Z, X′, X″, R, m and n are as defined above);and

deprotecting the carboxyl group at the β-position of the aspartic acid,the carboxyl group at the γ-position of the glutamic acid, the carbamidogroup at the β-position of asparagine or the carbamido group at theγ-position of glutamine of the cyclic depsipeptide.

The cyclic depsipeptides of the formula (1′) can be prepared bycondensing several synthesized oligopeptides followed byself-condensation, besides a process for the stepwise condensations ofamino acids followed by self-condensation according to the conventionalpeptide synthesis as mentioned above.

According to this alternative process, the cyclic depsipeptides can bealso prepared, for example, by the following steps comprising:

condensing the depsipeptide represented by the formula (9), which isobtained in the first half of the above-mentioned stepwisecondensations,

(wherein A, B, W, X, X′ and R are as defined above) with a tetrapeptide(or a tripeptide or a dipeptide) represented by the formula (19)(D)_(m)—(E)_(n)—F—Z(X″)—Y  (19)(wherein D, E, F, Z, X″, Y, m and n are as defined above), or condensinga depsipeptide represented by the formula (5)

(wherein A, X and R are as defined above) with a tripeptide (or adipeptide) represented by the formula (19′)B—W(X′)—(D′)_(m)—Y  (19′)(wherein B, D, W, X′, Y and m are as defined above), removing theamino-protecting group Y in the depsipeptide thus obtained, and furthercondensing with a tripeptide (or a dipeptide) represented by the formula(19″)(E)_(n)—F—Z(X″)—Y  (19″)(wherein E, F, Z, X″, Y and n are as defined above) to form thedepsipeptide represented by the above formula (16);

removing the amino-protecting group of the aspartic acid, asparagine,glutamic acid or glutamine in the depsipeptide to form a depsipeptiderepresented by the formula (17)

(wherein A, B, W, D, E, F, Z, X, X′, X″ and R are as defined above);

removing the carboxyl-protecting group X in the depsipeptide andsubsequent self-condensation to form a cyclic depsipeptide representedby the formula (18)

(wherein A, B, W, D, E, F, Z, X′, X″ and R are as defined above); and

deprotecting the carboxyl group at the β-position of the aspartic acid,the carboxyl group at the γ-position of the glutamic acid, the carbamidogroup at the β-position of asparagine or the carbamido group at theγ-position of glutamine of the cyclic depsipeptide.

The cyclic depsipeptides thus prepared may be converted, if required, topharmacologically acceptable salts thereof.

Any of the conventional processes adopted in peptide synthesis may beapplied in the above synthesis steps for preparing the cyclicdepsipeptides of the invention.

For instance, condensation reaction for forming a peptide bond includesa process using a condensing agent, azide process, a process using anacid anhydride, a process using an active ester, a process by redox, aprocess using an enzyme or the like.

Where peptide synthesis is to be carried out by the process using acondensing agent, there may be preferably employedN,N-dicyclohexyl-carbodiimide (hereinafter referred to as “DCC”) or1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, i.e.,water-soluble carbodiimide (hereinafter referred to as “WSCI”),O-(1H-benzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(hereinafter referred to as “TBTU”),benzotriazole-1-yl-oxy-tris(dimethylamino)-phosphoniumhexafluorophosphate (hereinafter referred to as “BOP”) and the like. Itis also preferable to simultaneously add an additive commonly employedfor preventing racemization such as N-hydroxy-succinimide,N-hydroxybenzotriazole (hereinafter referred to as “HOBt”),N-hydroxy-5-norbornene-2,3-dicarbodiimide-benzotriazole and the like.

Main condensing agents which may be employed in the azide processinclude diphenylphosphoryl azide (hereinafter referred to as “DPPA”),diethylphosphoryl cyanide and the like.

It is generally preferable to apply any known protection procedures tothe carboxyl group, amino group, ω-carbamido group and the like, whichdo not participate in the said condensation reaction.

The protecting group which may be used in the protection proceduresincludes, for example, a t-butoxycarbonyl (hereinafter referred to as“Boc”) group, a benzyloxycarbonyl group, a p-methoxy-benzyloxycarbonylgroup or a 9-fluorenylmethoxy-carbonyl (hereinafter referred to as“FMoc”) or the like as an amino-protecting group; a benzyloxy group or at-butoxy (hereinafter referred to as “OtBu”) or the like as acarboxyl-protecting group; 4,4-dimethoxy-benzhydryl (hereinafterreferred to as “Mbh”) group, a trityl group or the like as a terminalcarbamido-protecting group.

Removal of the protecting groups in the preparation of the cyclicdepsipeptide according to the present invention should be carried outwithout affecting the peptide linkage and it should be well chosendepending on the type of protecting groups used.

The solvent which may be employed in each peptide synthesis as depictedabove includes, for example, anhydrous or hydrous chloroform,dichloromethane, ethyl acetate, N,N-dimethylformamide (hereinafterreferred to as “DMF”), dimethyl sulfoxide, pyridine, dioxane,tetrahydrofuran (hereinafter referred to as “THF”), dimethoxyethane,acetonitrile and the like and they may be used in combination with twoor more thereof if necessary. The condensation reaction may be carriedout in a temperature range of from about −20 to 50° C. as usual

Peptide synthesis may be carried out according to any of the liquidphase method and the solid phase method, and a column method or a batchmethod may be also applicable.

The cyclic depsipeptides of the present invention may be converted by amethod known per se to pharmacologically acceptable salts thereof suchas metal salts, e.g. sodium, potassium or calcium salt, ammonium saltsor organic amine salts, e.g. triethylamine salts, and thesepharmacologically acceptable salts may be also used as an agent forpromoting the production of apolipoprotein E.

Illustrative examples of the 3-hydroxy-middle chain or -long chainaliphatic acid of the formula (2) as a starting material to be used forthe synthesis of cyclic depsipeptides of the above formula (1) may be3-hydroxy-capric acid, 3-hydroxy-lauric acid, 3-hydroxy-myristic acidand the like.

The 3-hydroxy-middle chain or -long chain aliphatic acid may be in anyof D-isomer, L-isomer and racemate forms.

One example of the synthesis routes of the cyclic depsipeptides of theinvention using 3-hydroxy-myristic acid as a starting material isillustrated by the following Reaction Scheme (SEQ ID NOS:1-7):

The cyclic depsipeptides of the invention may strongly promote theproduction of apolipoprotein E in Hep G2 cell that is theapolipoprotein-producing cell and therefore are useful as a therapeuticagent for neurologic damages and also as an antidementia agent.Moreover, they are useful for the treatment of disorders of peripheralnervous system such as diabetic neuropathy, disorders of peripheralnervous system caused by deficiency of Vitamin B group (B₁, B₂, B₁₂,etc.) and the like.

The cyclic depsipeptides or pharmacologically acceptable salts thereofaccording to the invention may greatly inhibit the production ofapolipoprotein B and promote the production of apolipoprotein A1, whilepromoting the production of apolipoprotein E as described above, andthey are then useful as an antihyperlipemic agent.

The cyclic depsipeptides or pharmacologically acceptable salts thereofaccording to the invention may be formulated to pharmaceuticalpreparations of various dosage forms. More specifically, suchpharmaceutical preparations may be, for example, solid preparations suchas tablets, hard capsules, soft capsules, granules, powders, etc. andliquid preparations such as solutions, emulsions, suspensions, etc. Thepreparations for parenteral administration may be injections,suppositories, etc.

In preparing such pharmaceutical preparations, conventional additivesmay be added, for example, excipients, stabilizers, antiseptics,solubilizers, wetting agents, emulsifying agents, lubricants, sweeteningagents, coloring agents, flavors, isotonic agents, buffering agents,antioxidants and the like.

As the additives, there may be mentioned, for example, starch, sucrose,fructose, lactose, glucose, mannitol, sorbitol, precipitated calciumcarbonate, crystalline cellulose, carboxymethylcellulose, dextrin,gelatin, acacia, magnesium stearate, talc, hydroxypropyl-methylcelluloseand the like.

Where the cyclic depsipeptides of the invention are to be applied in theform of solutions or injections, the cyclic depsipeptide orpharmacologically acceptable salt thereof as the active ingredient maybe dissolved or suspended in any conventional diluent. The diluent mayinclude, for example, a physiological saline, a Ringer's solution, anaqueous glucose solution, alcohols, fatty acid esters, glycols,glycerols, oils and fats derived from plant or animal sources, paraffinsand the like. These preparations may be prepared according to anyconventional method.

A usual clinical dose may be in the range of 0.5-5000 mg per day foradult when orally given. More preferably, it is in the range of 5-500mg.

A usual dose may be in the range of 0.05-5000 mg per day for adult whenparenterally administered.

Illustrative examples of the preparation of the present cyclicdepsipeptides will be explained hereinafter by way of SynthesisExamples, test examples for the promoting action of the present cyclicdepsipeptides on the production of apolipoprotein E will be given by wayof Examples, and examples of the pharmaceutical preparations containingas an active ingredient the present cyclic depsipeptides will beillustrated by way of Preparation Examples.

In the following Synthesis Examples and Examples, if the amino acidconstituting the depsipeptide or cyclic depsipeptide is in the form of aD-isomer, it will be specifically indicated, and unless otherwisespecified, the corresponding amino acid shall be in the form of anL-isomer.

SYNTHESIS EXAMPLE 1

To a solution of 3-hydroxymyristic acid (5.00 g) and triethylamine (2.85g) in DMF (50 ml) was added benzyl bromide (2.43 ml) and the mixture wasstirred at room temperature overnight. After the solvent was removed invacuo, to the residue were added ethyl acetate and water. The separatedethyl acetate layer was washed twice with water and then dried overanhydrous sodium sulfate. After the ethyl acetate was removed in vacuo,purification was carried out by a silica gel column chromatography(silica gel 30 g, chloroform:methanol=100:0-10) to afford 3.69 g ofbenzyl 3-hydroxymyristate.

(NMR data for benzyl 3-hydroxymyristate)

¹H-NMR (δ ppm, CDCl₃): 7.33-7.40 (5H, m), 5.16 (2H, s), 3.95-4.05 (1H,m), 2.85 (1H, d, J=4.4 Hz), 2.56 (1H, dd, J=2.9, 17 Hz), 2.46 (1H, dd,J=9.0, 17 Hz), 1.20-1.60 (20H, m), 0.88 (3H, t, J=6.8 Hz)

SYNTHESIS EXAMPLE 2

To a solution of benzyl 3-hydroxymyristate (3.00 g) obtained inSynthesis Example 1, Boc-isoleucine (2.22 g) and dimethylaminopyridine(77 mg) in dichloromethane (25 ml) was added dropwise under ice-coolinga solution of DDC (2.78 g) in dichloromethane (25 ml) and the mixturewas stirred under ice-cooling for one hour and then at room temperaturefor 2 hours. After a precipitate was filtered off, the dichloromethanewas removed in vacuo. The residue was dissolved in ethyl acetate andwashed in turn with 0.5N hydrochloric acid, 5% aqueous sodiumhydrogencarbonate and saturated aqueous sodium chloride, dried overanhydrous sodium sulfate. After the ethyl acetate was removed in vacuo,purification was carried out by a silica gel column chromatography(silica gel 100 g, hexane:ethyl acetate=200:0-15) to afford 4.62 g ofthe intermediate compound (1).

(NMR data for the intermediate compound (1)).

¹H-NMR (δ ppm, CDCl₃): 7.30-7.39 (5H, m), 5.24-5.33 (1H, m), 5.12 (1H,s), 5.10 (1H, d, J=2.5 Hz), 4.98-5.05 (1H, m), 4.15-4.25 (1H, m),2.56-2.72 (2H, m), 1.75-1.90 (1H, m), 1.50-1.70 (2H, m), 1.20-1.45 (19H,m), 1.44 (9H, s), 1.10-1.20 (1H, m), 0.86-0.93 (9H, m)

SYNTHESIS EXAMPLE 3

A solution of the intermediate compound (1) (4.62 g) obtained inSynthesis Example 2 in trifluoroacetic acid (hereinafter referred to as“TFA”) (9 ml) was stirred at room temperature for 15 minutes. After theTFA was removed in vacuo, the residue was dissolved in ethyl acetate andwashed with 5% aqueous sodium hydrogencarbonate. It was dried overanhydrous sodium sulfate and then the ethyl acetate was removed in vacuoto afford 3.78 g of the intermediate compound (2).

(NMR data for the intermediate compound (2))

¹H-NMR (δ ppm, CDCl₃): 7.31-7.39 (5H, m), 5.24-5.32 (1H, m), 5.06-5.14(2H, m), 3.02-3.29 (1H, m), 2.75-2.69 (2H, m), 1.50-1.75 (4H, m),1.30-1.40 (1H, m), 1.10-1.30 (20H, m), 0.85-0.94 (9H, m)

SYNTHESIS EXAMPLE 4

To a solution of the intermediate compound (2) (3.78 g) obtained inSynthesis Example 3, Boc-D-leucine· monohydrate (2.10 g) and HOBt·H₂O(1.25 g) in dichloromethane (50 ml) was added under ice-cooling WSCI(1.78 g). The solution was stirred under ice-cooling for one hour andthen at room temperature overnight. After the dichloromethane wasremoved in vacuo, to the residue was added ethyl acetate and washed inturn with IN hydrochloric acid, water, 5% aqueous sodiumhydrogencarbonate and water and then dried over anhydrous sodiumsulfate.

After the ethyl acetate was removed in vacuo, purification was carriedout by a silica gel column chromatography (silica gel 80 g, hexane:ethylacetate=200: 10-25) to afford 5.58 g of the intermediate compound (3).

(NMR Data for the Intermediate Compound (3))

¹H-NMR (δ ppm, CDCl₃): 7.30-7.39 (5H, m), 6.60-6.70 (1H, m), 5.25-5.30(1H, m), 5.07-5.14 (2H, m), 4.75-4.95 (1H, m), 4.45-4.55 (1H, m),4.10-4.20 (1H, m), 2.55-2.71 (2H, m), 1.80-1.95 (1H, m), 1.50-1.70 (3H,m), 1.35-1.50 (2H, m), 1.45 (9H, 2s), 1.20-1.35 (19H, m), 1.00-1.20 (1H,m), 0.85-0.95 (15H, m)

SYNTHESIS EXAMPLE 5

A solution of the intermediate compound (3) (8.77 g) obtained inSynthesis Example 4 in TFA (17 ml) was stirred at room temperature for45 minutes. After the TFA was removed in vacuo, the residue wasdissolved in ethyl acetate and washed with 5% aqueous sodiumhydrogencarbonate. It was dried over anhydrous sodium sulfate and theethyl acetate was removed in vacuo to afford the amine compound. To asolution of the amine compound thus obtained, Fmoc-L-aspartic acidβ-t-butyl ester (5.46 g) and HOBt·H₂O (2.24 g) in dichloromethane (80ml) was added under ice-cooling WSCI (2.80 g). This solution was stirredunder ice-cooling for 2 hours and then at room temperature overnight.After the dichloromethane was removed in vacuo, to the residue wereadded ethyl acetate and 10% aqueous citric acid. The separated ethylacetate layer was washed in turn with water, 5% aqueous sodiumhydrogencarbonate and water, and then dried over anhydrous sodiumsulfate. After the ethyl acetate was removed in vacuo, purification wascarried out by a silica gel column chromatography (silica gel 60 g,hexane:ethyl acetate=200: 10-50) to afford 11.06 g of the intermediatecompound (4).

(NMR Data for the Intermediate Compound (4))

¹H-NMR (δppm, CD₃OD): 7.78 (2H, d, J=7.8 Hz), 7.64 (2H, d, J=7.3 Hz),7.38 (2H, t, J=7.6 Hz), 7.25-7.35 (7H, m), 5.20-5.25 (1H, m), 5.07 (1H,s), 5.06 (1H, s), 4.35-4.50 (3H, m), 4.25-4.35 (2H, m), 4.15-4.25 (1H,m), 2.70-2.80 (1H, m), 2.55-2.65 (3H, m), 1.75-1.90 (1H, m), 1.50-1.75(4H, m), 1.44.(9H, s), 1.10-1.50 (21H, m), 0.75-0.95 (15H, m)

SYNTHESIS EXAMPLE 6

To a solution of the intermediate compound (4) (11.00 g) obtained inSynthesis Example 5 in DMF (125 ml) was added diethylamine (12.5 ml) andthe mixture was stirred at room temperature for 2 hours. After thesolvent was removed in vacuo, Fmoc-L-valine (3.91 g) andHOBt-monohydrate (1.94 g) were added and dissolved in dichloromethane(70 ml). To this solution was added under ice-cooling WSCI (2.43 g). Thesolution was stirred under ice-cooling for 2 hours and then at roomtemperature overnight. After the DMF was removed in vacuo, to theresidue were added ethyl acetate and 10% aqueous citric acid. Theseparated ethyl acetate layer was washed in turn with water, 5% aqueoussodium hydrogencarbonate and water and then dried over anhydrous sodiumsulfate. After the ethyl acetate was removed in vacuo, purification wascarried out by using a silica gel column chromatography (silica gel 100g, chloroform:methanol 200:0-10) to afford 10.65 g of the intermediatecompound (5).

(NMR Data for the Intermediate Compound (5))

¹H-NMR (δ ppm, CD₃OD): 7.66-7.82 (4H, m), 7.25-7.40 (9H, m), 5.15-5.25(1H, m), 5.00-5.10 (2H, m), 4.60-4.65 (1H, m), 4.30-4.50 (3H, m),4.20-4.30 (2H, m), 3.80 (1H, d, J=6.4 Hz), 2.90-3.00 (1H, m), 2.50-2.80(3H, m), 2.00-2.10 (1H, m), 1.60-1.95 (3H, m), 1.35-1.60 (3H, m), 1.43(9H, s), 1.10-1.35 (20H, m), 0.80-1.05 (21H, m)

SYNTHESIS EXAMPLE 7

To a solution of the intermediate compound (5) (2.74 g) obtained inSynthesis Example 6 in DMF (30 ml) was added diethylamine (3 ml) and themixture was stirred at room temperature for 4 hours. After the solventwas removed in vacuo, Fmoc-D-leucine (1.01 g) and HOBt-monohydrate (0.44g) were added and dissolved in dichloromethane (15 ml). To this solutionwas added under ice-cooling WSCI (0.55 g). The solution was stirredunder ice-cooling for 2 hours and then at room temperature overnight.After the DMF was removed in vacuo, to the residue were added ethylacetate and 10% aqueous citric acid. The separated ethyl acetate layerwas washed in turn with water, 5% aqueous sodium hydrogencarbonate andwater and then dried over anhydrous sodium sulfate. After the ethylacetate was removed in vacuo, purification was carried out by using asilica gel column chromatography (silica gel 30 g,chloroform:methanol=200:0-2) to afford 2.54 g of the intermediatecompound (6).

(NMR Data for the Intermediate Compound (6))

¹H-NMR (δ ppm, CD₃OD): 7.60-7.80 (4H, m), 7.25-7.40 (9H, m), 5.15-5.25(1H, m), 5.19 (2H, s), 4.50-4.55 (1H, m), 4.25-4.40 (4H, m), 4.15-4.25(2H, m), 4.13 (1H, d, J=5.9 Hz), 2.80-2.90 (1H, m), 2.50-2.80 (3H, m),2.15-2.25 (1H, m), 1.80-1.90 (1H, m), 1.50-1.75 (7H, m), 1.36 (9H, s),1.10-1.50 (21H, m), 0.80-1.00 (27H, m)

SYNTHESIS EXAMPLE 8

To a solution of the intermediate compound (6) (2.00 g) obtained inSynthesis Example 7 in DMF (20 ml) was added diethylamine (2 ml) and themixture was stirred at room temperature for 4 hours. After the solventwas removed in vacuo, Fmoc-L-leucine (0.67 g) and HOBt·monohydrate (0.29g) were added and dissolved in dichloromethane (20 ml). To this solutionwas added under ice-cooling WSCI (0.36 g). The solution was stirredunder ice-cooling for 2 hours and then at room temperature overnight.After the dichloromethane was removed in vacuo, to the residue wereadded chloroform and 10% aqueous citric acid. The separated chloroformlayer was washed in turn with water, 5% aqueous sodium hydrogencarbonateand water and then dried over anhydrous sodium sulfate. After thechloroform was removed in vacuo, purification was carried out by using asilica gel column chromatography (silica gel 25 g, chloroformmethanol=200:0-6) to afford 2.19 g of the intermediate compound (7).

(NMR Data for the Intermediate Compound (7))

¹H-NMR (δ ppm, CD₃OD): 7.75-7.80 (2H, m), 7.55-7.65 (2H, m), 7.25-7.40(9H, m), 5.20-5.30 (1H, m), 5.05-5.10 (2H, m), 4.15-4.65 (7H, m),4.00-4.10 (1H, m), 3.85-3.95 (1H, m), 2.90-3.00 (1H, m), 2.55-2.75 (3H,m), 2.05-2.15 (1H, m), 1.85-1.95 (1H, m), 1.50-1.80 (10H, m), 1.37, 1.39(9H, 2s), 1.10-1.50 (21H, m), 0.75-1.00 (33H, m)

SYNTHESIS EXAMPLE 9

To a solution of the intermediate compound (7) (2.00 g) obtained inSynthesis Example 8 in DMF (20 ml) was added diethylamine (2 ml) and themixture was stirred at room temperature for 2.5 hours. After the solventwas removed in vacuo, N-α-Fmoc-γ-Mbh-L-glutamine (1.02 g) andHOBt·monohydrate (0.26 g) were added and dissolved in a mixed solvent ofDMF (20 ml) and dichloromethane (10 ml). To this solution was addedunder ice-cooling WSCI (0.33 g). The solution was stirred underice-cooling for 2 hours and then at room temperature for 2 days. Afterthe mixed solvent was removed in vacuo, to the residue were addedchloroform and 10% aqueous citric acid. The separated chloroform layerwas washed in turn with water, 5% aqueous sodium hydrogencarbonate andwater and then dried over anhydrous sodium sulfate. After the chloroformwas removed in vacuo, purification was carried out by using a silica gelcolumn chromatography (silica gel 50 g, chloroform: methanol=200:0-4) toafford 1.92 g of the intermediate compound (8).

(NMR Data for the Intermediate Compound (8))

¹H-NMR (δ ppm, CD₃OD): 7.20-7.40 (10H, m), 7.13 (4H, d, J=8.3 Hz), 6.83(4H, dd, J=2.0, 8.8 Hz), 6.08 (1H, s), 5.15-5.25 (1H, m), 5.00-5.10 (4H,m), 4.70-4.80 (1H, m), 4.50-4.60 (1H, m), 4.30-4.50 (2H, m), 4.20-4.30(2H, m), 4.01 (1H, d, J=6.4 Hz), 3.76 (6H, s), 2.90-2.95 (1H, m),2.75-2.85 (1H, m), 2.60-2.70 (2H, m), 2.35-2.45 (2H, m), 2.10-2.20 (1H,m), 1.80-2.05 (3H, m), 1.50-1.80 (10H, m), 1.41 (9H, s), 1.10-1.50 (21H,m), 0.80-1.00 (33H, m)

SYNTHESIS EXAMPLE 10

To a solution of the intermediate compound (8) (1.85 g) obtained inSynthesis Example 9 in DMF (20 ml) was added diethylamine (2 ml) and themixture was stirred at room temperature for one hour. After the solventwas removed in vacuo, the residue was dissolved in methanol (60 ml), 5%palladium carbon (0.20 g) was added and the mixture was stirred underhydrogen atmosphere for 3 hours. The palladium carbon was filtered off.The methanol was removed in vacuo and then the crude product thusobtained was purified by a silica gel column chromatography (silica gel30 g, chloroform:methanol=200:0-30) to afford 1.16 g of the intermediatecompound (9).

(NMR Data for the Intermediate Compound (9))

¹H-NMR (δ ppm, CD₃OD): 7.14 (4H, d, J=8.3 Hz), 6.85 (4H, dd, J=2.2, 9.3Hz), 6.08 (1H, s), 5.23 (1H, quint., J=6.0 Hz), 4.79 (1H, dd, J=5.0, 8.8Hz), 4.46 (1H, dd, J=10, 21 Hz), 4.46 (1H, t, J=10 Hz), 4.28 (1H, d,J=6.8 Hz), 4.24 (1H, t, J=7.4 Hz), 4.02 (1H, d, J=6.4 Hz), 3.84 (1H, t,J=14 Hz), 3.77 (6H, 2s), 3.01 (1H, dd, J=5.2, 16 Hz), 2.75 (1H, dd,J=9.0, 16 Hz), 2.37-2.52 (3H, m), 2.34 (1H, dd, J=6.6, 15 Hz), 2.02-2.19(2H, m), 1.83-2.02 (2H, m), 1.54-1.83 (10H, m), 1.44 (9H, s), 1.08-1.53(21H, m), 0.81-1.08 (33H, m)

SYNTHESIS EXAMPLE 11

To a solution of the intermediate compound (9) (0.50 g) obtained inSynthesis Example 10 in DMF (200 ml) was added under ice-cooling DPPA(0.09 ml). Triethylamine (0.06 ml) was further added and the mixture wasstirred under ice-cooling for 3 hours and then at room temperatureovernight. After the solution was ice-cooled, DPPA (0.09 ml) andtriethylamine (0.06 ml) were added. The solution was stirred underice-cooling for 4 hours and then at room temperature for 3 days. Afterthe solvent was removed in vacuo, to the residue were added ethylacetate and 10% aqueous citric acid. The separated ethyl acetate layerwas washed in turn with water, 5% aqueous sodium hydrogen-carbonate andwater and then dried over anhydrous sodium sulfate. After the ethylacetate was removed in vacuo, purification was carried out by using asilica gel column chromatography (silica gel 25 g,chloroform:methanol=200:0-4) to afford 0.35 g of the cyclic depsipeptide(1) of the present invention.

(NMR Data for the Cyclic Depsipeptide (1))

¹H-NMR (δ ppm, CD₃OD): 7.07-7.20 (4H, m), 6.80-6.92 (4H, m), 6.01-6.10(1H, m), 5.14-5.29 (1H, m), 4.67-4.78 (1H, m), 4.21-4.45 (5H, m),4.06-4.16 (1H, m), 3.77 (3H, s), 3.76 (3H, s), 2.62-2.85 (2H, m),2.47-2.58 (1H, m), 2.21-2.44 (3H, m), 1.81-1.94 (4H, m), 1.53-1.81 (10H,m), 1.44 (9H, s), 1.08-1.53 (21H, m), 0.74-1.08 (33H, m)

SYNTHESIS EXAMPLE 12

A solution of the cyclic depsipeptide (1) obtained (0.35 g) in SynthesisExample 11 in TFA (5 ml) was stirred at room temperature for 1.5 hours.After the TFA was removed in vacuo, the residue was neutralized with 5%aqueous sodium hydrogencarbonate and extracted with a 10% methanolicsolution of chloroform. The chloroform layer was dried over anhydroussodium sulfate, the solvent was removed in vacuo and then purificationwas carried out by a silica gel column chromatography (silica gel 20 g,chloroform:methanol=100:0-50) to afford 0.23 g of the cyclicdepsipeptide (2) of the invention (hereinafter referred to as “Compound4”).

(NMR Data for Compound 4)

¹H-NMR (δ ppm, CD₃OD): 5.20-5.30 (1H, m), 4.80-4.90 (1H, m), 4.20-4.50(5H, m), 4.10-4.20 (1H, m), 2.80-2.95 (2H, m), 2.40-2.55 (2H, m),2.10-2.25 (2H, m), 1.80-2.00 (4H, m), 1.45-1.80 (10H, m), 1.10-1.45(21H, m), 0.70-1.10 (33H, m)

SYNTHESIS EXAMPLE 13 BzlO-Ala+D-Leu-Fmoc→Fmoc-D-Leu-Ala-OBzl

To L-alanine benzyl ester·p-toluenesulfonate (3.16 g) were added ethylacetate (100 ml) and 5% aqueous sodium hydrogencarbonate (50 ml), themixture was vigorously stirred and then allowed to stand. The separatedethyl acetate layer was dried over anhydrous sodium sulfate. After thesolvent was removed in vacuo, Fmoc-D-leucine (3.18 g) andHOBt·monohydrate (1.35 g) were added and dissolved in dichloromethane(100 ml). To this solution was added under ice-cooling WSCI (2.59 g).The solution was stirred under ice-cooling for one hour, allowed togradually rise up to room temperature and then stirred overnight. Thereaction solution was washed in turn with 10% aqueous citric acid,water, 5% aqueous sodium hydrogencarbonate and water and then dried overanhydrous sodium sulfate. After the solvent was removed in vacuo,purification was carried out by using a silica gel column chromatography(silica gel 50 g, chloroform:methanol:aqueous ammonia=20:1:0.05) toafford 5.36 g of the dipeptide (1).

(NMR Data for the Dipeptide (1))

¹H-NMR (δ ppm, CDCl₃) 7.76 (2H, d, J=7.8 Hz), 7.58 (2H, d, J=7.3 Hz),7.28-7.42 (9H, m), 6.57 (1H, d, J=6.3 Hz), 5.18 (1H, d, J=12.2 Hz), 5.12(1H, d, J=12.2 Hz), 5.09 (1H, d, J=8.3 Hz), 4.57-4.64 (1H, m), 4.41-4.46(2H, m), 4.20-4.23 (2H, m), 1.48-1.69 (3H, m), 1.41 (3H, d, J=6.8 Hz),0.93 (3H, d, J=6.8 Hz)

SYNTHESIS EXAMPLE 14 Fmoc-D-Leu-Ala-OBzl→Fmoc-Leu-D-Leu-Ala-OBzl

The dipeptide (1) (5.36 g) obtained in Synthesis Example 13 wasdissolved in DMF (50 ml), diethylamine (5 ml) was added and then themixture was stirred at room temperature for 2 hours. After the solventwas removed in vacuo, Fmoc-L-leucine (3.18 g) and HOBt·monohydrate (1.35g) were added and dissolved in dichloromethane (100 ml). To thissolution was added under ice-cooling WSCI (2.59 g). The solution wasstirred under ice-cooling for one hour, allowed to gradually rise up toroom temperature and then stirred overnight. The reaction solution waswashed in turn with 10% aqueous citric acid, water, 5% aqueous sodiumhydrogencarbonate and water and then dried over anhydrous sodiumsulfate. After the solvent was removed in vacuo, purification wascarried out by using a silica gel column chromatography (silica gel 50g, chloroform:methanol:aqueous ammonia=50:1:0.05) to afford 7.18 g ofthe tripeptide (1).

(NMR Data for the Tripeptide (1))

¹H-NMR (δ ppm, CDCl₃) 7.75 (2H, d, J=7.3 Hz), 7.53-7.57 (2H, m),7.28-7.41 (9H, m), 7.01 (1H, d, J=6.8 Hz), 6.43 (1H, d, J=8.3 Hz), 5.20(1H, d, J=6.8 Hz), 5.12 (1H, d, J=12.2 Hz), 5.05 (1H, d, J12.2 Hz),4.06-4.56 (6H, m), 1.47-1.78 (6H, m), 1.34 (3H, d, J=7.3 Hz), 0.90-0.95(12H, 25 m)

SYNTHESIS EXAMPLE 15 Fmoc-Leu-D-Leu-Ala-OBzl→Leu-D-Leu-Ala-OBzl

The tripeptide (1) (7.08 g) obtained in Synthesis Example 14 wasdissolved in DMF (70 ml), diethylamine (7 ml) was added and then themixture was stirred at room temperature for 2 hours. After the solventwas removed in vacuo, purification was carried out by using a silica gelcolumn chromatography (silica gel 100 g, chloroform methanol:aqueousammonia=50:1:0.05 to 10:1 0.05) to afford 2.08 g of the tripeptide (2).

(NMR Data for the Tripeptide (2))

¹H-NMR (δ ppm, CDCl₃) 7.57 (1H, d, J=8.3 Hz), 7.31-7.38 (5H, m), 6.86(1H, d, J=7.3 Hz), 5.19 (1H, d, J=12.2 Hz), 5.11 (1H, d, J=12.2 Hz),4.52-4.59 (1H, m), 4.41-4.47 (1H, m), 3.38 (1H, dd, J=3.9, 9.8 Hz),1.28-1.79 (6H, m), 1.40 (3H, d, J=6.8 Hz), 0.91-0.96 (12H, m)

SYNTHESIS EXAMPLE 16 Leu-D-Leu-Ala-OBzl→Fmoc-Gln(Mbh)-Leu-D-Leu-Ala-OBzl

The tripeptide (2) (2.08 g) obtained in Synthesis Example 15,N-α-Fmoc-N-γ-Mbh-L-glutamine (3.05 g) and HOBt·monohydrate (0.76 g) weredissolved in a mixed solvent of dichloromethane (30 ml) and DMF (3 ml).To this solution was added under ice-cooling WSCI (1.47 g). The solutionwas stirred under ice-cooling for one hour, allowed to gradually rise upto room temperature and then stirred overnight. The reaction solutionwas diluted with chloroform (100 ml) and washed in turn with 10% aqueouscitric acid, water, 5% aqueous sodium hydrogencarbonate and water andthen dried over anhydrous sodium sulfate. The solvent was removed invacuo to afford 5.02 g of the tetrapeptide (1).

(NMR Data for the Tetrapeptide (1))

¹H-NMR (δ ppm, d-DMSO) 8.52 (1H, d, J=8.8 Hz), 7.27-8.13 (18H, m),7.08-7.15 (4H, m), 6.83-6.85 (4H, m), 6.01 (1H, d, J=8.3 Hz), 5.06 (1H,d, J=12.7 Hz), 5.11 (1H, d, J=12.7 Hz), 4.01-4.27 (7H, m), 3.70 (6H, s),2.23-2.29 (2H, m), 1.79-1.92 (2H, m), 1.46-1.58 (6H, m), 1.29 (3H, d,J=7.3 Hz), 0.78-0.87 (12H, m)

SYNTHESIS EXAMPLE 17Fmoc-Gln(Mbh)-Leu-D-Leu-Ala-OBzl→Fmoc-Gln(Mbh)-Leu-D-Leu-Ala(Mbh)-Fmoc

The tetrapeptide (1) (1.50 g) obtained in Synthesis Example 16 wasdissolved in a mixed solvent of methanol (150 ml) and DMF (75 ml), 10%palladium carbon was added and the mixture was stirred under hydrogenatmosphere for 2 hours. After the palladium carbon was filtered off, thesolvent was removed in vacuo to afford 1.43 g of the tetrapeptide (2).

(NMR Data for the Tetrapeptide (2))

¹H-NMR (δ ppm, d-DMSO) 8.56 (1H, d, J=8.8 Hz), 7.28-8.17 (12H, m),7.13-7.16 (4H, m), 6.82-6.85 (4H, m), 6.01 (1H, d, J=8.3 Hz), 4.09-4.27(7H, m), 3.71 (6H, s), 2.24-2.33 (2H, m), 1.75-1.92 (2H, m), 1.44-1.59(6H, m), 1.24 (3H, d, J=7.3 Hz), 0.78-0.87 (12H, m)

SYNTHESIS EXAMPLE 18 Val-OBzl→D-Leu-Val-OBzl

To L-valine benzyl ester·p-toluenesulfonate (4.56 g) were added ethylacetate (100 ml) and 5% aqueous sodium hydrogencarbonate (50 ml), themixture was vigorously stirred and then allowed to stand. The separatedethyl acetate layer was dried over anhydrous sodium sulfate. After thesolvent was removed in vacuo, Fmoc-D-leucine (4.24 g) andHOBt·monohydrate (1.78 g) were added and dissolved in dichloromethane(40 ml). To this solution was added under ice-cooling WSCI (3.46 g). Thesolution was stirred under ice-cooling for one hour, allowed togradually rise up to room temperature and then stirred overnight. Thereaction solution was diluted with chloroform (80 ml) and washed withsaturated aqueous sodium chloride and then dried over anhydrous sodiumsulfate. After the solvent was removed in vacuo, the resulting residuewas dissolved in DMF (50 ml) and diethylamine (8 ml) was added thereto.The mixture was stirred at room temperature for 3 hours. After thesolvent was removed in vacuo, purification was carried out by using asilica gel column chromatography (silica gel 75 g,chloroform:methanol=100:0-2) to afford 3.27 g of the dipeptide (2).

(NMR Data for the Dipeptide (2))

¹H-NMR (δ ppm, CD₃OD) 7.29-7.39 (5H, m), 5.20 (1H, d, J=12.2 Hz), 5.14(1H, d, J=12.2 Hz), 4.35 (1H, d, J=5.9 Hz), 3.42 (1H, dd, J=6.4, 7.8Hz), 2.13-2.22 (1H, m), 1.63-1.73 (1H, m), 1.49-1.55 (1H, m), 1.33-1.40(1H, m), 0.91-0.95 (12H, m)

SYNTHESIS EXAMPLE 19 D-Leu-Val-OBzl→Ala-D-Leu-Val-OBzl

The dipeptide (2) (2.88 g) obtained in Synthesis Example 18,Fmoc-L-alanine (1.97 g) and HOBt·monohydrate (0.89 g) were dissolved ina mixed solvent of DMF (25 ml) and THF (25 ml), and WSCI (1.72 g) wasadded under ice-cooling. The solution was stirred under ice-cooling forone hour, allowed to gradually rise up to room temperature and thenstirred overnight. After the solvent was removed lie in vacua, theresidue was dissolved in DMF (20 ml) and diethylamine (3 ml) was addedand the mixture was stirred at room temperature for one hour. After thesolvent was removed in vacuo, purification was carried out by using asilica gel column chromatography (silica gel 50 g,chloroform:methanol=100:0-2) to afford 1.59 g of the tripeptide (3).

(NMR Data for the Tripeptide (3))

¹H-NMR (δ ppm, CDCl₃) 7.57 (1H, d, J=8.3 Hz), 7.30-7.38 (5H, m), 6.86(1H, d, J=8.3 Hz), 5.18 (1H, d, J=12.2 Hz), 5.11 (1H, d, J12.2 Hz),4.43-4.52 (2H, m), 3.49 (1H, q, J=6.8 Hz), 2.14-2.25 (1H, m), 1.74-1.80(1H, m), 1.62-1.70 (1H, m), 1.53-1.60 (1H, m), 1.51 (2H, br), 1.32 (3H,d, J=6.8 Hz), 0.86-0.96 (12H, m)

SYNTHESIS EXAMPLE 20 Ala-D-Leu-Val-OBzl→Fmoc-Gln(Mbh)-Ala-D-Leu-Val-OBzl

To a solution of the tripeptide (3) (1.59 g) obtained in SynthesisExample 19, N-α-Fmoc-N-γ-Mbh-L-glutamine (2.41 g) and HOBt·monohydrate(0.60 g) in DMF (50 ml) was added under ice-cooling WSCI (1.17 g). Themixture was stirred under ice-cooling for one hour, allowed to graduallyrise up to room temperature and then stirred overnight. To the reactionsolution was added water (100 ml). The insolubles precipitated out werefiltered off and dried to afford 3.78 g of the tetrapeptide (3).

(NMR Data for the Tetrapeptide (3))

¹H-NMR (δ ppm, d-DMSO) 8.51 (1H, d, J=8.3 Hz), 8.07 (1H, d, J=8.3 Hz),7.91-7.99 (2H, m), 7.86 (2H, d, J=7.3 Hz), 7.67-7.72 (2H, m), 7.43 (2H,d, J=7.8 Hz), 7.28-7.41 (9H, m), 7.13-7.15 (4H, m), 6.82-6.85 (4H, m),6.01 (1H, d, J=8.8 Hz), 5.13 (1H, d, J=12.7 Hz), 5.07 (1H, d, J=12.7Hz), 3.71-4.44 (7H, m), 3.71 (6H, s), 2.26-2.53 (4H, m), 2.02-2.08 (1H,m), 1.45-1.93 (3H, m), 1.21 (3H, d, J=7.3 Hz), 0.80-0.86 (12H, m)

SYNTHESIS EXAMPLE 21Fmoc-Gln(Mbh)-Ala-D-Leu-Val-OBzl→Fmoc-Gln(Mbh)-Ala-D-Leu-Val

The tetrapeptide (3) (1.45 g) obtained in Synthesis Example 20 wasdissolved in a mixed solvent of methanol (70 ml) and DMF (70 ml), 10%palladium carbon (0.5 g) was added and the mixture was stirred underhydrogen atmosphere for one hour. After the palladium carbon wasfiltered off, the solvent was removed in vacuo to afford 1.76 g of thetetrapeptide (4).

(NMR Data for the Tetrapeptide (4))

¹H-NMR (δ ppm, d-DMSO) 8.53 (1H, d, J=8.3 Hz), 7.28-7.97 (12H, m),7.13-7.16 (4H, m), 6.82-6.86 (4H, m), 6.01 (1H, d, J=8.8 Hz), 4.02-4.43(7H, m), 3.71 (6H, s), 2.23-2.39 (1H, m), 1.79-2.06 (3H, m), 1.45-1.60(3H, m), 1.21 (3H, d, J=6.8 Hz), 0.81-0.86 (12H, m)

SYNTHESIS EXAMPLE 22 Val-OBzl→D-Ala-Val-OBzl

To L-valine benzyl ester·p-toluenesulfonate (2.28 g) were added ethylacetate (100 ml) and 5% aqueous sodium hydrogencarbonate (50 ml), themixture was vigorously stirred and then allowed to stand. The separatedethyl acetate layer was dried over anhydrous sodium sulfate. After thesolvent was removed in vacuo, Fmoc-D-leucine (1.87 g) andHOBt·monohydrate (0.89 g) were added and dissolved in dichloromethane(20 ml). To this solution was added under ice-cooling WSCI (1.73 g). Thesolution was stirred under ice-cooling for one hour, allowed togradually rise up to room temperature and then stirred overnight. Thereaction solution was diluted with chloroform (30 ml) and washed withsaturated aqueous sodium chloride and dried over anhydrous sodiumsulfate. After the solvent was removed in vacuo, the residue wasdissolved in DMF (30 ml), diethylamine (3 ml) was added and the mixturewas stirred at room temperature for one hour. After the solvent wasremoved in vacuo, purification was carried out by using a silica gelcolumn chromatography (silica gel 50 g, chloroform:methanol=100:0-2) toafford 1.47 g of the dipeptide (3).

(NMR Data for the Dipeptide (3))

¹H-NMR (δ ppm, CDCl₃) 7.71 (1H, d, J=8.8 Hz), 7.31-7.39 (5H, m), 5.21(1H, d, J=12.2 Hz), 5.12 (1H, d, J=12.2 Hz), 4.56 (1H, dd, J=4.4, 8.8Hz), 3.53 (1H, q, J=6.8 Hz), 2.17-2.28 (1H, m), 1.50 (2H, br), 1.33 (1H,d, J=6.8 Hz), 0.93 (3H, d, J=6.8 Hz), 0.88 (3H, d, J=6.8 Hz)

SYNTHESIS EXAMPLE 23 D-Ala-Val-OBzl→Leu-D-Ala-Val-OBzl

To a solution of the dipeptide (3) (1.47 g) obtained in SynthesisExample 22, Fmoc-L-leucine (1.98 g) and HOBt·monohydrate (0.83 g) in DMF(20 ml) was added under ice-cooling WSCI (1.61 g). The mixture wasstirred under ice-cooling for one hour, allowed to gradually rise up toroom temperature and then stirred overnight. The reaction solution wasdiluted with ethyl acetate (50 ml) and washed in turn with 5% aqueoussodium hydrogencarbonate, water, 10% aqueous citric acid and water andthen dried over anhydrous sodium sulfate. After the solvent was removedin vacuo, the residue was dissolved in DMF (30 ml), diethylamine (3 ml)was added and the mixture was stirred at room temperature for one hour.After the solvent was removed in vacuo, purification was carried out byusing a silica gel column chromatography (silica gel 50 g,chloroform−chloroform:methanol:aqueous ammonia=50:1:0.05) to afford 2.11g of the tripeptide (4).

(NMR Data for the Tripeptide (4))

¹H-NMR (δ ppm, CDCl₃) 7.66 (1H, d, J=7.3 Hz), 7.30-7.38 (5H, m), 6.90(1H, d, J=8.3 Hz), 5.19 (1H, d, J=12.7 Hz), 5.10 (1H, d, J=12.7 Hz),4.49-4.57 (2H, m), 3.37 (1H, dd, J=3.4, 9.8 Hz), 2.14-2.26 (1H, m), 1.40(1H, d, J=6.8 Hz), 1.29-1.77 (5H, m), 0.85-0.96 (12H, m)

SYNTHESIS EXAMPLE 24 Leu-D-Ala-Val-OBzl→Fmoc-Gln(Mbh)-Leu-D-Ala-Val-OBzl

To a solution of the tripeptide (4) (2.11 g) obtained in SynthesisExample 23, N-α-Fmoc-N-γ-Mbh-L-glutamine (3.20 g) and HOBt·monohydrate(0.80 g) in DMF (25 ml) was added under ice-cooling WSCI (1.55 g). Themixture was stirred under ice-cooling for one hour, allowed to graduallyrise up to room temperature and then stirred overnight. To the reactionsolution was added water (100 ml). The insolubles thus precipitated outwere filtered off and dried to afford 4.38 g of the tetrapeptide (5).

(NMR Data for the Tetrapeptide (5))

¹H-NMR (δ ppm, d-DMSO) 8.51 (1H, d, J=8.3 Hz), 7.87-7.99 (3H, m), 7.86(2H, d, J=7.3 Hz), 7.67-7.69 (2H, m ), 7.28-7.44 (10H, m), 7.12-7.15(4H, m), 6.83-6.85 (4H, m), 6.01 (1H, d, J=8.3 Hz), 5.14 (1H, d, J12.2Hz), 5.08 (1H, d, J=12.2 Hz), 4.02-4.40 (7H, m), 3.71 (6H, s), 1.46-2.26(8H, m), 1.19 (3H, d, J=6.8 Hz), 0.80-0.86 (12H, m)

SYNTHESIS EXAMPLE 25Fmoc-Gln(Mbh)-Leu-D-Ala-Val-OBzl→Fmoc-Gln(Mbh)-Leu-D-Ala-Val

The tetrapeptide (5) (1.45 g) obtained in Synthesis Example 24 wasdissolved in a mixed solvent of methanol (100 ml) and DMF (50 ml), 10%palladium carbon (0.50 g) was added and the mixture was stirred underhydrogen atmosphere for 1.5 hours. After the palladium carbon wasfiltered off, the solvent was removed in vacuo to afford 2.00 g of thetetrapeptide (6).

(NMR Data for the Tetrapeptide (6))

¹H-NMR (δ ppm, CD₃OD) 7.62-7.78 (4H, m), 7.28-7.39 (4H, m), 7.12-7.15(4H, m), 6.83-6.86 (4H, m), 6.09 (1H, s), 3.87-4.45 (7H, m), 3.75 (6H,s), 1.33-2.39 (8H, m), 0.85-0.97 (15H, m)

SYNTHESIS EXAMPLE 26Fmoc-Gln(Mbh)-Leu-D-Leu-Val-OBzl→Fmoc-Gln(Mbh)-Leu-D-1S, Leu-Val

The tetrapeptide (5) (1.01 g) obtained in Synthesis Example 24 wasdissolved in a mixed solvent of methanol (80 ml) and DMF (80 ml), 10%palladium carbon (0.55 g) was added and the mixture was stirred underhydrogen atmosphere for 2 hours. After the palladium carbon was filteredoff, the solvent was removed in vacuo to afford 1.98 g of thetetrapeptide (7).

(NMR Data for the Tetrapeptide (7))

¹H-NMR (δ ppm, d-DMSO) 8.55 (1H, d, J=8.8 Hz), 7.28-8.01 (12H, m),7.13-7.16 (4H, m), 6.82-6.85 (4H, m), 6.01 (1H, d, J=8.8 Hz), 4.02-4.33(7H, m), 3.71 (6H, s), 2.25-2.29 (2H, m), 1.77-2.05 (3H, m), 1.45-1.60(6H, m), 0.80-0.88 (18H, m)

SYNTHESIS EXAMPLE 27

To a solution of benzyl 3-hydroxymyristate (1.67 g),Fmoc-L-alanine-monohydrate (1.65 g) and dimethylamino-pyridine (60 mg)in dichloromethane (120 ml) was added under ice-cooling DCC (1.54 g) andthe mixture was stirred under ice-cooling for one hour, allowed togradually rise up to room temperature and then stirred overnight. Afterthe precipitate was filtered off, the dichloromethane was removed invacuo. To the residue was added ethyl acetate (30 ml), the insolubleswere filtered off and then the ethyl acetate was removed in vacuo. Theresidue was dissolved in DMF (20 ml), diethylamine (2 ml) was added andthen the mixture was stirred at room temperature for one hour. After thesolvent was removed in vacuo, purification was carried out by using asilica gel column chromatography (silica gel 50 g,chloroform:methanol=200:0-10) to afford 1.70 g of the intermediatecompound (10).

(NMR Data for the Intermediate Compound (10))

¹H-NMR (δ ppm, CDCl₃) 7.32-7.36 (5H, m), 5.22-5.29 (1H, m), 5.10-5.11(2H, m), 3.34-3.44 (1H, m), 2.60-2.63 (2H, m), 1.20-1.59 (25H, m), 0.88(3H, t, J=7 Hz)

SYNTHESIS EXAMPLE 28

To a solution of the intermediate compound (10) (1.70 g) obtained inSynthesis Example 27, Fmoc-D-leucine (1.48 g) and HOBt·monohydrate (0.62g) in dichloromethane (50 ml) was added under ice-cooling WSCI (1.20 g).The mixture was stirred under ice-cooling for one hour, allowed togradually rise up to room temperature and then stirred overnight. Thereaction solution was washed with saturated aqueous sodium chloride anddried over anhydrous sodium sulfate. After the solvent was removed invacuo, the residue was dissolved in DMF (20 ml), diethylamine (3 ml) wasadded and the mixture was stirred at room temperature for one hour.After the solvent was removed in vacuo, purification was carried out byusing a silica gel column chromatography (silica gel 75 g,chloroform:methanol=200:0-10) to afford 1.93 g of the intermediatecompound (11).

(NMR Data for the Intermediate Compound (11))

¹H-NMR (δ ppm, CDCl₃) 7.65 (1H, d, J=7.3 Hz), 7.32-7.38 (5H, m),5.25-5.31 (1H, m), 5.07-5.14 (2H, m), 4.48-4.53 (1H, m), 3.35-3.38 (1H,m), 2.61-2.71 (2H, m), 1.24-1.74 (28H, m), 0.94 (3H, d, J=11.2 Hz), 0.93(3H, d, J=11.2 Hz), 0.88 (3H, t, J=6.8 Hz)

SYNTHESIS EXAMPLE 29

To a solution of the intermediate compound (11) (1.93 g) obtained inSynthesis Example 28, Fmoc-L-aspartic acid β-t-butyl ester (1.53 g) andHOBt·monohydrate (0.55 g) in dichloromethane (100 ml) was added underice-cooling WSCI (1.07 g). The mixture was stirred under ice-cooling forone hour, allowed to gradually rise up to room temperature and thenstirred overnight. The reaction solution was washed with saturatedaqueous sodium chloride and dried over anhydrous sodium sulfate. Afterthe solvent was removed in vacuo, the residue was dissolved in DMF (30ml), diethylamine (3 ml) was added and the mixture was stirred at roomtemperature for one hour. After the solvent was removed in vacuo,purification was carried out by using a silica gel column chromatography(silica gel 75 g, chloroform:methanol=200:0-10) to afford 2.76 g of theintermediate compound (12).

(NMR Data for the Intermediate Compound (12))

¹H-NMR (δ ppm, CDCl₃) 7.78 (1H, d, J=8.8 Hz), 7.09 (1H, d, J=7.3 Hz),7.32-7.35 (5H, m), 5.23-5.26 (1H, m), 5.09-5.14 (2H, m), 4.45-4.53 (2H,m), 3.58-3.61 (1H, m), 2.60-2.75 (4H, m), 1.24-1.90 (34H, m), 1.43 (9H,s), 0.88 (3H, t, J=6.8 Hz)

SYNTHESIS EXAMPLE 30

To a solution of the intermediate compound (12) (0.79 g) obtained inSynthesis Example 29, the tetrapeptide (9) (1.08 g) obtained asdescribed in the following Synthesis Example 71 and HOBt·monohydrate(0.17 g) in DMF (25 ml) was added under ice-cooling WSCI (0.33 g). Themixture was stirred under ice-cooling for one hour, allowed to graduallyrise up to room temperature and then stirred overnight. The reactionsolution was diluted with dichloromethane (100 ml) and washed withsaturated aqueous sodium chloride and then dried over anhydrous sodiumsulfate. After the solvent was removed in vacuo, the residue wasdissolved in DMF (30 ml), diethylamine (3 ml) was added and the mixturewas stirred at room temperature for one hour. After the solvent wasremoved in vacuo, purification was carried out by using a silica gelcolumn chromatography (silica gel 75 g, chloroform:methanol aqueousammonia=50:1:0.05 to 20:1:0.05) to afford 1.46 g of the intermediatecompound (13).

(NMR Data for the Intermediate Compound (13))

¹H-NMR (δ ppm, CD₃OD) 7.29-7.35 (5H, m), 7.11-7.14 (4H, m), 6.83-6.86(4H, m), 6.07 (1H, s), 5.21-5.25 (1H, m), 5.11 (2H, s), 4.00-4.64 (7H,m), 3.76 (6H, s), 2.64-2.98 (4H, m), 1.55-2.34 (14H, m), 1.44 (9H, s),1.25-1.31 (23H, m), 0.87-0.98 (27H, m)

SYNTHESIS EXAMPLE 31

To a solution of the intermediate compound (13) (1.46 g) obtained inSynthesis Example 30 in methanol (150 ml) was added 10% palladium carbon(0.44 g) and the mixture was stirred under hydrogen atmosphere for 30minutes. The palladium carbon was filtered off and the methanol wasremoved in vacuo. Then, the residue (0.70 g) was dissolved in a mixedsolvent of THF (390 ml) and DMF (130 ml) and then cesium chloride (0.94g), potassium chloride (0.37 g), N-methylmorpholine (0.11 g),HOBt·monohydrate (0.30 g) and WSCI (0.96 g) were in turn added. Themixture was stirred at room temperature for 5 days. The reactionsolution was diluted with ethyl acetate (400 ml) and washed in turn withwater, 5% aqueous sodium hydrogencarbonate, water, 10% aqueous citricacid and water and then dried over anhydrous sodium sulfate. After thesolvent was removed in vacuo, the residue was applied to a silica gelcolumn (silica gel, 75 g) and the fractions eluted withchloroform:methanol=40:1 (500 ml) were concentrated. The residue (0.91g) was dissolved in TFA (5 ml) and the solution was stirred at roomtemperature for 2 hours. After the solvent was removed in vacuo, theresidue was neutralized with 5% aqueous sodium hydrogencarbonate andextracted with a 10% methanolic solution of chloroform. The organiclayer was dried over anhydrous sodium sulfate and then purified by asilica gel column chromatography (silica gel 30 g, chloroformmethanol=10:1 to 4:1) to afford 0.31 g of the cyclic depsipeptide (3) ofthe invention (hereinafter referred to as “Compound 5”).

(NMR Data for Compound 5)

¹H-NMR (δ ppm, d-DMSO) 6.65-9.24 (9H, m), 4.98-5.10 (1H, m), 4.04-4.44(7H, m), 2.50-2.66 (2H, m), 1.98-2.42 (4H, m), 1.74-1.90 (2H, m),1.23-1.63 (30H, m), 0.74-0.90 (30H, m)

FAB-MS 979 (MH⁺), 1017 (MK⁺)

SYNTHESIS EXAMPLE 32

To a solution of benzyl 3-hydroxymyristate (3.18 g), Fmoc-L-isoleucine(3.70 g) and dimethylaminopyridine (0.10 g) in dichloromethane (250 ml)was added under ice-cooling DCC (3.14 g) and the mixture was stirredunder ice-cooling for one hour and then allowed to gradually rise up toroom temperature and stirred overnight. After a precipitate was filteredoff, the dichloromethane was removed in vacuo. To the residue was addedethyl acetate (30 ml), insolubles were filtered off and the ethylacetate was removed in vacuo. The residue was purified by a silica gelcolumn chromatography (silica gel 200 g, chloroform) to afford 7.14 g ofthe intermediate compound (14).

(NMR Data for the Intermediate Compound (14))

¹H-NMR (δ ppm, CDCl₃) 7.29-7.77 (13H, m), 5.27-5.33 (2H, m), 5.11 (2H,s), 4.29-4.34 (3H, m), 4.23 (1H, t, J=6.8 Hz), 2.57-2.72 (2H, m),1.07-1.90 (23H, m), 0.86-0.95 (9H, m)

SYNTHESIS EXAMPLE 33

To a solution of the intermediate compound (14) (7.14 g) obtained inSynthesis Example 32 in DMF (70 ml) was added diethylamine (7 ml) andthe mixture was stirred at room temperature for 5 hours. After thesolvent was removed in vacuo, purification was carried out by a silicagel column chromatography (silica gel 200 g, chloroformmethanol=200:0-10) to afford 4.33 g of the intermediate compound (15).

(NMR Data for the Intermediate Compound (15))

¹H-NMR (δ ppm, CDCl₃) 7.33-7.36 (5H, m), 5.24-5.31 (1H, m), 5.11 (2H,s), 3.19 (1H, d, J=4.9 Hz), 2.57-2.66 (2H, m), 1.05-1.75 (25H, m),0.86-0.94 (9H, m)

SYNTHESIS EXAMPLE 34

To a solution of the intermediate compound (15) (1.83 g) obtained inSynthesis Example 33, Fmoc-D-alanine (0.91 g) and HOBt·monohydrate (0.40g) in dichloromethane (70 ml) was added under ice-cooling WSCI (0.77 g)and the mixture was stirred under ice-cooling for 3.5 hours. Thereaction solution was washed with a saturated aqueous solution of sodiumchloride and dried over anhydrous sodium sulfate. After the solvent wasremoved in vacuo, the residue was dissolved in DMF (50 ml), diethylamine(4 ml) was added and the mixture was stirred at room temperature for 4hours. After the solvent was removed in vacuo, the residue was dissolvedin ethyl acetate (100 ml), washed with a saturated aqueous solution ofsodium chloride and dried over anhydrous sodium sulfate. After thesolvent was removed in vacuo, the residue was purified by a silica gelcolumn chromatography (silica gel 100 g, chloroform methanol:aqueousammonia=40:1:0.05) to afford 1.77 g of the intermediate compound (16).

(NMR Data for the Intermediate Compound (16))

¹H-NMR (δ ppm, CDCl₃) 7.66 (1H, d, J=8.B Hz), 7.29-7.38 (5H, m),5.26-5.31 (1H, m), 5.13 (1H, d, J=12.2 Hz), 5.08 (1H, d, J=12.2 Hz),4.49-4.54 (1H, m), 3.48-3.53 (1H, m), 2.56-2.72 (2H, m), 1.04-1.95 (25H,m), 0.86-0.93 (12H, m)

SYNTHESIS EXAMPLE 35

To a solution of the intermediate compound (16) (1.77 g) obtained inSynthesis Example 34, Fmoc-L-aspartic acid β-t-butyl ester (1.21 g) andHOBt·monohydrate (0.40 g) in dichloromethane (100 ml) was added underice-cooling WSCI (0.77 g) and the mixture was stirred under ice-coolingfor 5.5 hours. The reaction solution was washed with a saturated aqueoussolution of sodium chloride and dried over anhydrous sodium sulfate.After the solvent was removed in vacuo, the residue was dissolved in DMF(30 ml), diethylamine (3 ml) was added and the mixture was stirred atroom temperature for 2 hours. After the solvent was removed in vacuo,the residue was purified by a silica gel chromatography (silica gel 100g, chloroform:methanol:aqueous ammonia=40:1:0.05) to afford 2.33 g ofthe intermediate compound (17). To a solution of this intermediatecompound (17) (0.77 g), the tetrapeptide (9) (1.46 g) obtained in thefollowing Synthesis Example 71 and HOBt·monohydrate (0.15 g) in DMF (50ml) was added under ice-cooling WSCI (0.29 g) and the mixture wasstirred under ice-cooling for one hour, allowed to gradually rise up toa room temperature and stirred overnight. The reaction solution wasdiluted with ethyl acetate (200 ml), washed in turn with water, 10%aqueous citric acid, water and 5% aqueous sodium hydrogencarbonate andthen dried over anhydrous sodium sulfate. After the solvent was removedin vacuo, the residue was dissolved in DMF (40 ml), diethylamine (1 ml)was added and the mixture was stirred at room temperature for one hour.After the solvent was removed in vacuo, the residue was purified by asilica gel column chromatography (silica gel 100 g,chloroform:methanol:aqueous ammonia=50:1:0.1 to 20:1:0.1) to afford 0.73g of the intermediate compound (18). The intermediate compound (18)(0.73 g) was dissolved in methanol (100 ml), 10% palladium carbon (0.27g) was added and the mixture was stirred under hydrogen atmosphere forone hour. The palladium carbon was filtered off and the methanol wasremoved in vacuo to afford 0.65 g of the intermediate compound (19).

(NMR Data for the Intermediate Compound (19))

¹H-NMR (δ ppm, CDCl₃) 6.65-8.20 (1SH, m), 6.11 (1H, d, J=6.3 Hz),5.26-5.30 (1H, m), 3.93-4.53 (6H, m), 3.76 (6H, s), 1.10-3.03 (40H, m),0.86-0.94 (30H, m)

SYNTHESIS EXAMPLE 36

The intermediate compound (19) (0.65 g) obtained in Synthesis Example 35was dissolved in a mixed solvent of THF (390 ml) and DMF (130 ml) andthen cesium chloride (0.94 g), potassium chloride (0.37 g),N-methyl-morpholine (0.11 g), HOBt·monohydrate (0.30 g) and WSCI (0.96g) were in turn added. The mixture was stirred at room temperature for 5days. The reaction solution was diluted with ethyl acetate (400 ml) andwashed in turn with water, 5% aqueous sodium hydrogencarbonate, water,10% aqueous citric acid and water and then dried over anhydrous sodiumsulfate. After the solvent was removed in vacuo, the residue was appliedto a silica gel column (silica gel, 100 g) and the fractions eluted withchloroform:methanol=20:1 (500 ml) were concentrated. The residue (1.05g) was dissolved in TFA (5 ml) and the solution was stirred at roomtemperature for 2 hours. After the solvent was removed in vacuo, theresidue was neutralized with 5% aqueous sodium hydrogencarbonate andextracted with a 10% methanolic solution of chloroform. The organiclayer was dried over anhydrous sodium sulfate and then purified by asilica gel column chromatography (silica gel 30 g,chloroform:methanol=10:1 to 4:1) to afford 0.24 g of the cyclicdepsipeptide (4) of the invention.

(NMR Data for the Cyclic Depsipeptide (4))

¹H-NMR (δ ppm, d-DMSO) 6.63-9.86 (9H, m), 4.94-5.14 (1H, 25 m),3.72-4.55 (7H, m), 1.04-2.66 (38H, m), 0.74-0.91 (30H, m)

FAB-MS 979 (MH^(+),) 1017 (MK⁺)

SYNTHESIS EXAMPLE 37

To a solution of the intermediate compound (4) (8.37 g) obtained inSynthesis Example 5 in DMF (80 ml) was added diethylamine (8 ml) and themixture was stirred at room temperature for 1.5 hours. After the solventwas removed in vacuo, the residue was dissolved in ethyl acetate (150ml), washed with a saturated aqueous solution of sodium sulfate anddried over anhydrous sodium sulfate. After the solvent was removed invacuo, purification was carried out by a silica gel columnchromatography (silica gel 200 g, chloroform:methanol=30:0-1) to afford5.81 g of the intermediate compound (20).

(NMR Data for the Intermediate Compound (20))

¹H-NMR (δ ppm, CDCl₃) 7.28-7.37 (6H, m), 6.88 (1H, d, J=6.9 Hz),5.23-5.28 (1H, m), 5.06-5.13 (2H, m), 4.45-4.83 (2H, m), 3.62-3.66 (1H,m), 2.55-2.85 (4H, m), 1.24-1.94 (37H, m), 0.85-1.07 (15H, m)

SYNTHESIS EXAMPLE 38

To a solution of the intermediate compound (20) (1.12 g) obtained inSynthesis Example 37, the tetrapeptide (2) obtained in Synthesis Example17 (1.43 g) and HOBt·monohydrate (0.44 g) in DMF (50 ml) was added underice-cooling WSCI (0.44 g) and the mixture was stirred under ice-coolingfor one hour, allowed to gradually rise up to room temperature andstirred overnight. The reaction solution was diluted with chloroform(200 ml), washed in turn with 10% aqueous citric acid, water, 5% aqueoussodium hydrogencarbonate and water and then dried over anhydrous sodiumsulfate. After the solvent was removed in vacuo, the residue wasdissolved in DMF (15 ml), diethylamine (0.7 ml) was added and themixture was stirred at room temperature for one hour. After the solventwas removed in vacuo, the residue was purified by a silica gel columnchromatography (silica gel 75 g, chloroform:methanol=100: 1-5) to afford0.94 g of the intermediate compound (21).

(NMR Data for the Intermediate Compound (21))

¹H-NMR (δ ppm, CDCl₃) 6.35-7.80 (20H, m), 6.28 (1H, d, J=8.3 Hz),5.20-5.24 (1H, m), 5.06-5.11 (2H, m), 3.90-4.65 (6H, m), 3.78 (3H, s),3.77 (3H, s), 3.18-3.22 (1H, m), 2.22-2.65 (8H, m), 1.21-1.95 (43H, m),0.79-0.97 (30H, m)

SYNTHESIS EXAMPLE 39

To a solution of the intermediate compound (21) (0.93 g) obtained inSynthesis Example 38 in methanol (150 ml) was added 10% palladium carbon(0.31 g) and the mixture was stirred under hydrogen atmosphere for onehour. The palladium carbon was filtered off and the methanol was removedin vacuo to afford 0.77 g of the intermediate compound (22). Then, theintermediate compound (22) (0.50 g) was dissolved in THF (95 ml) andN-methylmorpholine (0.08 g) and HOBt·monohydrate (0.25 g) were added toform Solution A. In a mixed solvent of THF (190 ml) and DMF (95 ml) wereadded in turn cesium chloride (0.70 g), potassium chloride (0.28 g) andWSCI (0.71 g) to form Solution B. Solution A was added dropwise toSolution B over 20 minutes while stirring at room temperature and themixture was stirred at room temperature for 5 days. The reactionsolution was diluted with ethyl acetate (200 ml) and washed in turn withwater, 5% aqueous sodium hydrogencarbonate, water, 10% aqueous citricacid and water and then dried over anhydrous sodium sulfate. After thesolvent was removed in vacuo, the residue was applied to a silica gelcolumn (silica gel, 100 g) and the fractions eluted withchloroform:methanol=50:1 (500 ml) were concentrated. The residue (0.79g) was dissolved in TFA (3 ml) and the solution was stirred at roomtemperature for 1.5 hours. After the solvent was removed in vacuo, theresidue was neutralized with 5% aqueous sodium hydrogencarbonate andextracted with a 10% methanolic solution of chloroform. The organiclayer was dried over anhydrous sodium sulfate and then purified by asilica gel column chromatography (silica gel 30 g,chloroform:methanol=10:1 to 4:1) to afford 0.21 g of the cyclicdepsipeptide (5) of the invention.

(NMR Data for the Cyclic Depsipeptide (5))

¹H-NMR (δ ppm, d-DMSO) 6.63-9.74 (9H, m), 4.92-5.11 (1H, m), 3.81-4.56(7H, m), 1.75-2.69 (8H, m), 1.21-1.57 (32H, m), 0.77-0.87 (30H, m)

FAB-MS 993 (MH⁺), 1031 (MK⁺)

SYNTHESIS EXAMPLE 40

To a solution of the intermediate compound (20) (1.55 g) obtained inSynthesis Example 37, the tetrapeptide (6) obtained in Synthesis Example25 (2.00 g) and HOBt·monohydrate (0.22 g) in DMF (40 ml) was added underice-cooling WSCI (0.43 g) and the mixture was stirred under ice-coolingfor one hour, allowed to gradually rise up to room temperature andstirred overnight. The reaction solution was diluted with chloroform(200 ml), washed with a saturated aqueous solution of sodium chlorideand then dried over anhydrous sodium sulfate. After the solvent wasremoved in vacuo, the residue was dissolved in DMF (50 ml), diethylamine(3 ml) was added and the mixture was stirred at room temperature for onehour. After the solvent was removed in vacuo, the residue was purifiedby a silica gel column chromatography (silica gel 75 g,chloroform−chloroform:methanol:aqueous ammonia=50:1:0.1 to 20:1:0.1) toafford 1.21 g of the intermediate compound (23).

(NMR Data for the Intermediate Compound (23))

¹H-NMR (δ ppm, CDCl₃) 6.60-7.68 (20H, m), 6.12 (1H, d, J=8.4 Hz),5.20-5.24 (1H, m), 5.06-5.11 (2H, m), 3.85-4.48 (6H, m), 3.79 (6H, s),3.32-3.36 (1H, m), 2.25-2.52 (8H, m), 1.25-2.06 (41H, m), 0.86-0.99(30H, m)

SYNTHESIS EXAMPLE 41

To a solution of the intermediate compound (23) (1.21 g) obtained inSynthesis Example 40 in methanol (100 ml) was added 10% palladium carbon(0.33 g) and the mixture was stirred under hydrogen atmosphere for onehour. The palladium carbon was filtered off and the methanol was removedin vacuo to afford 0.88 g of the intermediate compound (24). Then, theintermediate compound (24) (0.65 g) was dissolved in a mixed solvent ofTHF (390 ml) and DMF (130 ml), and to the solution were added in turncesium chloride (0.94 g), potassium chloride (0.37 g),N-methylmorpholine (0.11 g), HOBt·monohydrate (0.30 g) and WSCI (0.96 g)and the mixture was stirred at room temperature for 5 days. The reactionsolution was diluted with ethyl acetate (400 ml) and washed in turn withwater, 5% aqueous sodium hydrogencarbonate, water, 10% aqueous citricacid and water and then dried over anhydrous sodium sulfate. After thesolvent was removed in vacuo, the residue was applied to a silica gelcolumn (silica gel, 75 g) and the fractions eluted withchloroform:methanol=40:1 (500 ml) were concentrated. The residue (1.01g) was dissolved in TFA (5 ml) and the solution was stirred at roomtemperature for 2 hours. After the solvent was removed in vacuo, theresidue was neutralized with 5% aqueous sodium hydrogencarbonate andextracted with a 10% methanolic solution of chloroform. The organiclayer was dried over anhydrous sodium sulfate and then purified by asilica gel column chromatography (silica gel 30 g,chloroform:methanol=10:0-3) to afford 0.40 g of the cyclic depsipeptide(6) of the invention.

(NMR Data for the Cyclic Depsipeptide (6))

¹H-NMR (δ ppm, d-DMSO) 6.63-10.01 (9H, m), 4.93-5.11 (1H, m), 3.99-4.60(7H, m), 2.61-2.78 (2H, m), 2.00-2.34 (4H, m), 1.80-1.98 (2H, m),1.16-1.76 (30H, m), 0.76-0.91 (30H, m)

FAB-MS 979 (MH⁺), 1017 (MK⁺)

SYNTHESIS EXAMPLE 42

To a solution of the intermediate compound (20) (1.10 g) obtained inSynthesis Example 37, the tetrapeptide (4) obtained in Synthesis Example21 (1.76 g) and HOBt·monohydrate (0.22 g) in DMF (25 ml) was added underice-cooling WSCI (0.43 g) and the mixture was stirred under ice-coolingfor one hour, allowed to gradually rise up to room temperature andstirred overnight. The reaction solution was diluted with chloroform(200 ml), washed with a saturated aqueous solution of sodium chlorideand then dried over anhydrous sodium sulfate. After the solvent wasremoved in vacuo, the residue was dissolved in DMF (50 ml), diethylamine(3 ml) was added and the mixture was stirred at room temperature for onehour. After the solvent was removed in vacuo, the residue was purifiedby a silica gel column chromatography (silica gel 75 g,chloroform−chloroform:methanol:aqueous ammonia=50:1:0.1) to afford 1.22g of the intermediate compound (25).

(NMR Data for the Intermediate Compound (25))

¹H-NMR (δ ppm, CDCl₃) 8.28-8.31 (1H, m), 6.74-7.50 (19H, m), 6.18 (1H,d, J=8.3 Hz), 5.19-5.24 (1H, m), 5.09 (2H, s), 4.37-4.49 (4H, m),3.93-4.06 (2H, m), 3.28-3.31 (1H, m), 3.02-3.10 (1H, m), 2.55-2.71 (4H,m), 2.18-2.46 (3H, m), 1.10-2.10 (41H, m), 0.83-0.97 (30H, m)

SYNTHESIS EXAMPLE 43

To a solution of the intermediate compound (25) (1.22 g) obtained inSynthesis Example 42 in methanol (150 ml) was added 10% palladium carbon(0.18 g) and the mixture was stirred under hydrogen atmosphere for onehour. The palladium carbon was filtered off and the methanol was removedin vacuo to afford 1.00 g of the intermediate compound (26). Then, theintermediate compound (26) (0.70 g) was dissolved in a mixed solvent ofTHF (390 ml) and DMF (130 ml), and to the solution were added in turncesium chloride (0.94 g), potassium chloride (0.37 g),N-methylmorpholine (0.11 g), HOBt·monohydrate (0.30 g) and WSCI (0.96 g)and the mixture was stirred at room temperature for 5 days. The reactionsolution was diluted with ethyl acetate (400 ml) and washed in turn withwater, aqueous sodium hydrogencarbonate, water, 10% aqueous citric acidand water and then dried over anhydrous sodium sulfate. After thesolvent was removed in vacuo, the residue was applied to a silica gelcolumn (silica gel, 75 g) and the fractions eluted withchloroform:methanol=40:1 (500 ml) were concentrated. The residue (1.10g) was dissolved in TFA (5 ml) and the solution was stirred at roomtemperature for 2 hours. After the solvent was removed in vacuo, theresidue was neutralized with 5% aqueous sodium hydrogencarbonate andextracted with a 10% methanolic solution of chloroform. The organiclayer was dried over anhydrous sodium sulfate and then purified by asilica gel column chromatography (silica gel 30 g, chloroformmethanol=10:0-3) to afford 0.38 g of the cyclic depsipeptide (7).

(NMR Data for the Cyclic Depsipeptide (7))

¹H-NMR (δ ppm, d-DMSO) 6.65-9.20 (9H, m), 4.95-5.09 (1H, m), 3.85-4.53(7H, m), 2.61-2.66 (1H, m), 2.34-2.37 (1H, m), 25 1.75-2.12 (6H, m),1.18-1.56 (30H, m), 0.78-0.88 (30H, m)

FAB-MS 979 (MH⁺), 1017 (MK⁺)

SYNTHESIS EXAMPLE 44

To a solution of the intermediate compound (20) (4.15 g) obtained inSynthesis Example 37, Fmoc-L-valine (1.85 g) and HOBt·monohydrate (0.81g) in dichloromethane (50 ml) was added under ice-cooling WSCI (1.56 g)and the mixture was stirred under ice-cooling for one hour and thenallowed to gradually rise up to room temperature and stirred overnight.The reaction solution was washed with a saturated aqueous solution ofsodium chloride and dried over anhydrous sodium sulfate. After thesolvent was removed in vacuo, the residue was dissolved in DMF (30 ml),diethylamine (3 ml) was added and the mixture was stirred at roomtemperature for 2 hours. The solvent was removed in vacuo and theresidue was purified by a silica gel column chromatography (silica gel100 g, chloroform−chloroform methanol=40:1) to afford 2.74 g of theintermediate compound (27).

(NMR Data for the Intermediate Compound (27))

¹H-NMR (δ ppm, CDCl₃) 8.30 (1H, t, J=8.8 Hz), 7.30-7.37 (5H, m), 7.08(1H, t, J=7.3 Hz), 6.75 (1H, d, J=8.3 Hz), 5.21-5.24 (1H, m), 5.11 (2H;s), 4.76-4.79 (1H, m), 4.44-4.50 (2H, m), 3.32-3.35 (1H, m), 2.55-2.71(4H, m), 2.23-2.26 (1H, m), 1.04-1.81 (28H, m), 0.82-0.99 (21H, m)

SYNTHESIS EXAMPLE 45

To a solution of the intermediate compound (27) (2.74 g) obtained inSynthesis Example 44, Fmoc-L-leucine (1.16 g) and HOBt·monohydrate (0.49g) in dichloromethane (50 ml) was added under ice-cooling WSCI (0.95 g)and the mixture was stirred under ice-cooling for one hour, allowed togradually rise up to room temperature and stirred overnight. Thereaction solution was washed with a saturated aqueous solution of sodiumchloride and then dried over anhydrous sodium sulfate. After the solventwas removed in vacuo, the residue was dissolved in DMF (30 ml),diethylamine (3 ml) was added and the mixture was stirred at roomtemperature for 4 hours. The solvent was removed in vacuo,Fmoc-L-leucine (1.16 g) and HOBt·monohydrate (0.49 g) were added to theresulting residue, the mixture was dissolved in dichloromethane (50 ml)and WSCI (0.95 g) was added under ice-cooling. The mixture was stirredunder ice-cooling for one hour, allowed to gradually rise up to roomtemperature and stirred overnight. The reaction solution was washed witha saturated aqueous solution of sodium chloride and then dried overanhydrous sodium sulfate. The residue was dissolved in DMF (30 ml),diethylamine (3 ml) was added and the mixture was stirred at roomtemperature for one hour. After the solvent was removed in vacuo, theresidue was purified by a silica gel column chromatography (silica gel75 g, chloroform−chloroform:methanol=40:1) to afford 2.15 g of theintermediate compound (28).

(NMR Data for the Intermediate Compound (28))

¹H-NMR (δ ppm, CDCl₃) 6.85-7.90 (10H, m), 5.20-5.25 (1H, m), 5.07-5.13(2H, m), 4.05-4.78 (5H, m), 3.28-3.40 (1H, m), 2.59-2.87 (4H, m),1.23-2.21 (44H, m), 0.84-0.96 (33H, m)

SYNTHESIS EXAMPLE 46

To a solution of the intermediate compound (28) (2.15 g) obtained inSynthesis Example 45, N-α-Fmoc-N-γ-Mbh-L-glutamine (1.21 g) andHOBt·monohydrate (0.30 g) in a mixed solvent of dichloromethane (25 ml)and DMF (25 ml) was added under ice-cooling WSCI (0.58 g) and themixture was stirred under ice-cooling for one hour and then allowed togradually rise up to room temperature and stirred overnight. Thereaction solution was concentrated, the residue was dissolved in DMF (30ml), diethylamine (5 ml) was added and the mixture was stirred at roomtemperature for one hour. After the solvent was removed in vacuo, theresidue was purified by a silica gel column chromatography (silica gel150 g, chloroform:methanol=30:0-1) to afford 2.92 g of the intermediatecompound (29).

(NMR Data for the Intermediate Compound (29))

¹H-NMR (δ ppm, CDCl₃) 6.82-7.75 (20H, m), 5.15-5.20 (1H, m), 5.07 (2H,s), 4.05-4.50 (6H, m), 3.78 (3H, s), 3.77 (3H, s), 3.31-3.35 (1H, m).,2.33-2.80 (8H, m), 1.22-2.08 (44H, m), 0.81-0.94 (33H, m)

SYNTHESIS EXAMPLE 47

To a solution of the intermediate compound (29) (2.92 g) obtained inSynthesis Example 46 in a mixed solvent of methanol (75 ml) and DMF (50ml) was added 10% palladium carbon (0.43 g) and the mixture was stirredunder hydrogen atmosphere for 1.5 hours. The palladium carbon wasfiltered off and the solvent was removed in vacuo to afford 2.77 g ofthe intermediate compound (30). Then, the intermediate compound (30)(2.14 g) was dissolved in a mixed solvent of DMF (390 ml) and THF (1170ml), and to the solution were added in turn cesium chloride (2.82 g),potassium chloride (1.11 g), N-methylmorpholine (0.33 g),HOBt·monohydrate (0.90 g) and WSCI (2.88 g) and the mixture was stirredat room temperature for 5 days. The reaction solution was concentrated,the residue was dissolved in ethyl acetate (400 ml), washed with water(200 ml) and dried over anhydrous sodium sulfate. After the solvent wasremoved in vacuo, the residue was purified by a silica gel column(silica gel 75 g, chloroform:ethyl acetate=1:1) to afford 1.04 g of thecyclic depsipeptide (8′). The cyclic depsipeptide (8′) (0.54 g) wasdissolved in TFA (5 ml) and the solution was stirred at room temperaturefor 2 hours.

After the solvent was removed in vacuo, the residue was neutralized with5% aqueous sodium hydrogencarbonate and extracted with a 10% methanolicsolution of chloroform. The organic layer was dried over anhydroussodium sulfate and then purified by a silica gel column chromatography(silica gel 30 g, chloroform:methanol=10:0-2) to afford 0.26 g of thecyclic depsipeptide (8) of the invention (hereinafter referred to as“Compound 6”).

(NMR Data for Compound 6)

¹H-NMR (d ppm, d-DMSO) 6.64-9.45 (9H, m), 5.04-5.14 (1H, m), 3.74-4.45(7H, m), 2.32-2.44 (2H, m), 2.05-2.20 (4H, m), 1.70-1.99 (2H, m),1.23-1.60 (33H, m), 0.82-0.94 (33H, m)

FAB-MS 1021 (MH⁺), 1059 (MK⁺)

SYNTHESIS EXAMPLE 48 Fmoc-Asp(OtBu)+Leu-OBzl→Fmoc-Asp(OtBu)-Leu

The dipeptide (6.63 g) was obtained from L-leucine benzylester·p-toluenesulfonate (6.00 g) and L-aspartic acid β-t-butyl ester(5.23 g) in the same manner as in Synthesis Example 13. The dipeptidethus obtained was debenzylated in the same manner as in SynthesisExample 17 to afford the desired dipeptide (5.21 g).

¹H-NMR (CDCl₃) 5 ppm: 7.75 (2H, d, J=7.8 Hz), 7.56 (2H, d, J=7.3 Hz),7.39 (2H, t, J=7.6 Hz), 7.30 (2H, dt, J=1.0, 7.3 Hz), 7.00 (1H, d, J=7.8Hz), 6.03 (1H, d, J=8.3 Hz), 5.60 (1H, br s), 4.52-4.64 (2H, m), 4.41(2H, d, J=6.8 Hz), 4.21 (1H, t, J=6.8 Hz), 2.87 (1H, dd, J=4.4, 17 Hz),2.62 (1H, dd, J=6.8, 17 Hz), 1.66-1.76 (2H, m), 1.55-1.65 (1H, m), 1.44(9H, s), 0.92 (6H, t, J=5.6 Hz)

SYNTHESIS EXAMPLE 49Fmoc-Gln(Mbh)-Leu-D-Leu+Val-Asp(OtBu)-D-Leu-OBzl→Fmoc-Gln(Mbh)-Leu-D-Leu-Val-Asp(OtBu)-D-Leu-OBzl

To a solution of the tripeptide (9) (0.92 g) obtained in the followingSynthesis Example 77, the tripeptide (6) (0.50 g) obtained in thefollowing Synthesis Example 73 and HOBt·monohydrate (0.17 g) in DMF (10ml) was added under ice-cooling WSCI (0.21 g). The solution was stirredunder ice-cooling for 2 hours and then stirred at room temperatureovernight. After the DMF was removed in vacuo, to the residue were addeda 10% methanolic solution of chloroform and a 10% aqueous solution ofcitric acid. The separated organic layer was washed in turn with water,5% aqueous sodium hydrogencarbonate and water and then dried overanhydrous sodium sulfate. After the solvent was removed in vacuo, theresidue was purified by a silica gel column chromatography (silica gel30 g, chloroform:ethyl acetate=100:0 to 85:15) and then solidified withdiethyl ether to afford 0.82 g of the desired hexapeptide (1).

¹H-NMR (DMSO-d₆) δ ppm: 8.53 (1H, d, J=8.3 Hz), 8.09 (1H, d, J=8.3 Hz),8.05 (1H, d, J=7.3 Hz), 8.01 (1H, d, J=7.8 Hz), 7.86 (2H, d, J=7.3 Hz),7.80-7.89 (1H, m), 7.64-7.76 (3H, m), 7.46 (1H, d, J=7.8 Hz), 7.39 (2H,t, J=7.3 Hz), 7.24-7.36 (7H, m), 7.13 (4H, d, J=8.8 Hz), 6.83 (4H, dd,J=2.0, 8.8 Hz), 6.00 (1H, d, J=8.8 Hz), 5.09 (2H, s), 4.65 (1H, dd,J=8.3, 14 Hz), 4.11-4.40 (7H, m), 3.98-4.07 (1H, m), 3.71 (3H, s), 3.70(3H, s), 2.63 (1H, dd, J=5.6, 16 Hz), 2.45-2.51 (1H, m), 2.19-2.34 (2H,m), 1.86-2.00 (2H, m), 1.73-1.85 (1H, m), 1.38-1.64 (9H, m), 1.33 (9H,m), 0.69-0.88 (24H, m)

SYNTHESIS EXAMPLE 50

The desired intermediate compound (5.55 g) was obtained using(R)-3-hydroxymyristic acid in the same manner as in Synthesis Example 32and the alcohol (2.77 g) obtained in the same manner as in SynthesisExample 1.

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=7.3 Hz), 7.59 (2H, d, J=5.4 Hz),7.39 (2H, t, J=7.3 Hz), 7.26-7.36 (7H, m), 5.24-5.34 (2H, m), 5.10(2H,), 4.35-4.43 (2H, m), 4.30 (1H, dd, J=4.6, 3.8 Hz), 4.22 (1H, t,J=7.1 Hz), 2.69 (1H, dd, J=7.3, 16 Hz), 2.59 (1H, dd, J5.4, 16 Hz),1.84-1.94 (1H, m), 1.54-1.69 (2H, m), 1.07-1.45 (20H, m), 0.81-0.97 (9H,m)

SYNTHESIS EXAMPLE 51

To a solution of the intermediate compound (5.45 g) obtained inSynthesis Example 50 in DMF (80 ml) was added diethylamine (8 ml) andthe mixture was stirred at room temperature for 1.5 hours. After thesolvent was removed in vacuo, the residue was purified by a silica gelcolumn chromatography (silica gel 50 g, chloroform:methanol=200:0-10) toafford 3.62 g of an amine derivative. To a solution of the aminederivative thus obtained, the dipeptide obtained in Synthesis Example 48(5.06 g) and HOBt·monohydrate (1.48 g) in dichloromethane (60 ml) wasadded under ice-cooling WSCI (1.85 g). The solution was stirred underice-cooling for 2 hours and then at room temperature overnight. Afterthe dichloromethane was removed in vacuo, ethyl acetate and a 10%aqueous solution of citric acid were added to the residue. The separatedethyl acetate layer was washed in turn with water, 5% aqueous sodiumhydrogencarbonate and water and then dried over anhydrous sodiumsulfate. After the ethyl acetate was removed in vacuo, the residue waspurified by a silica gel column chromatography (silica gel 100 g,hexane:ethyl acetate=200: 50-90) and then solidified with a solventsystem of diethyl ether-hexane to afford 7.11 g of the desiredintermediate compound.

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=7.3 Hz), 7.58 (2H, d, J=7.3 Hz),7.40 (2H, t, J=7.6 Hz), 7.28-7.37 (7H, m), 6.85 (1H, d, J=8.3 Hz), 6.49(1H, d, J=8.3 Hz), 5.97 (1H, d, J=8.3 Hz), 5.27 (1H, qui., J=6.2 Hz),5.12 (1H, d, J=13 Hz), 5.09 (1H, d, J=13 Hz), 4.51 (1H, dd, J=4.4, 8.8Hz), 4.47-4.56 (1H, m), 4.37-4.46 (3H, m), 4.22 (1H, t, J=6.8 Hz), 2.92(1H, dd, J=4.4, 17 Hz), 2.68 (1H, dd, J=6.8, 16 Hz), 2.58 (1H, dd,J=5.9, 16 Hz), 2.55-2.67 (1H, m), 1.75-1.88 (1H, m), 1.52-1.73 (5H, m),1.45 (9H, s), 1.07-1.46 (20H, m), 0.82-0.97 (15H, m)

SYNTHESIS EXAMPLE 52 (1)

To a solution of the intermediate compound (2.90 g) obtained inSynthesis Example 51 in DMF (30 ml) was added diethylamine (3 ml) andthe mixture was stirred at room temperature for 1.5 hours. After thesolvent was removed in vacuo, the residue was purified by a silica gelcolumn chromatography (silica gel 30 g, chloroform:methanol=100:0-7) toafford 2.22 g of an amine derivative. To a solution of the aminederivative thus obtained, Fmoc-L-valine (1.24 g) and HOBt·monohydrate(0.56 g) in dichloromethane (25 ml) was added under ice-cooling WSCI(0.70 g). The solution was stirred under ice-cooling for 2 hours andthen at room temperature for 2 days. After the dichloromethane wasremoved in vacuo, chloroform and a 10% aqueous solution of citric acidwere added to the residue. The separated chloroform layer was washed inturn with water, 5% aqueous sodium hydrogen-carbonate and water and thendried over anhydrous sodium sulfate. After the chloroform was removed invacuo, the residue was purified by a silica gel column chromatography(silica gel 30 g, chloroform:methanol=100:0-2) to afford 2.29 g of thedesired intermediate compound.

¹H-NMR (CDCl₃) δ ppm: 7.77 (2H, d, J=7.3 Hz), 7.59 (2H, dd, J=.9, 7.3Hz), 7.40 (2H, t, J=7.3 Hz), 7.28-7.37 (8H, m), 7.05 (1H, d, J=7.8 Hz),6.59 (1H, d, J=7.8 Hz), 5.30 (1H, d, J=7.3 Hz), 5.26 (1H, qui., J=6.3Hz), 5.12 (1H, d, J=14 Hz), 5.09 (1H, d, J=12 Hz), 4.68-4.75 (1H, m),4.45-4.52 (2H, m), 4.35-4.44 (2H, m), 4.23 (1H, t, J=6.8 Hz), 4.00 (1H,t, J=5.6 Hz), 2.92 (1H, dd, J=3.7, 17 Hz), 2.70 (1H, dd, J=6.8, 16 Hz),2.59 (1H, dd, J=6.6, 17 Hz), 2.58 (1H, dd, J=6.6, 17 Hz), 2.13-2.23 (1H,m), 1.85-1.94 (1H, m), 1.48-1.76 (5H, m), 1.41 (9H, s), 1.09-1.45 (20H,m), 0.99 (3H, d, J=6.8 Hz), 0.95 (3H, d, J=6.4 Hz), 25 0.80-0.92 (15H,m)

SYNTHESIS EXAMPLE 52 (2)

To a solution of the intermediate compound (1.58 g) obtained inSynthesis Example 52 (1) in DMF (15 ml) was added diethylamine (1.5 ml)and the mixture was stirred at room temperature for 1.5 hours. After thesolvent was removed in vacuo, the residue was purified by a silica gelcolumn chromatography (silica gel 25 g, chloroform methanol=100:0-8) toafford 1.09 g of an amine derivative. To a solution of the aminederivative thus obtained, the tripeptide (9) (1.29 g) obtained in thefollowing Synthesis Example 77 and HOBt·monohydrate (0.24 g) in DMF (20ml) was added under ice-cooling WSCI (0.30 g). The solution was stirredunder ice-cooling for 2 hours and then at room temperature overnight.After the solvent was removed in vacuo, chloroform and a 10% aqueoussolution of citric acid were added to the residue. The separatedchloroform layer was washed in turn with water, 5% aqueous sodiumhydrogencarbonate and water and then dried over anhydrous sodiumsulfate. After the chloroform was removed in vacuo, the residue waspurified by a silica gel column chromatography (silica gel 50 g,chloroform:methanol=200:0-6) to afford 1.91 g of the desiredintermediate compound.

¹H-NMR (CDCl₃) δ ppm: 7.73 (2H, d, J=7.3 Hz), 7.63 (1H, d, J=8.3 Hz),7.59 (1H, d, J=8.8 Hz), 7.55 (2H, d, J=7.3 Hz), 7.38 (2H, t, J=7.6 Hz),7.20-7.35 (8H, m), 7.18 (2H, d, J=8.3 Hz), 7.14 (2H, d, J=8.8 Hz), 6.84(4H, dd, J=4.2, 8.3 Hz), 6.60-7.10 (4H, m), 6.40 (1H, d, J=B.3 Hz), 6.24(1H, d, J=8.3 Hz), 5.18 (1H, qui., J=6.3 Hz), 5.07 (2H, s), 4.78-4.85(1H, m), 4.45-4.55 (2H, m), 4.25-4.44 (4H, m), 4.10-4.20 (3H, m), 3.77(3H, s), 3.74 (3H, s), 2.89 (1H, dd, J=7.3, 16 Hz), 2.71 (1H, dd, J=5.1,17 Hz), 2.61 (1H, dd, J=6.8, 16 Hz), 2.51 (1H, dd, J=6.4, 16 Hz),2.14-2.32 (3H, m), 1.90-2.11 (2H, m), 1.76-1.87 (3H, m), 1.43 (9H, s),1.05-1.65 (29H, m), 0.95 (3H, d, J=6.3 Hz), 0.79-0.93 (24H, m), 0.76(3H, d, J=5.4 Hz), 0.74 (3H, d, J=5.9 Hz)

SYNTHESIS EXAMPLE 52 (3)

To a solution of the intermediate compound (1.90 g) obtained inSynthesis Example 52 (2) in DMF (15 ml) was added diethylamine (1.5 ml)and the mixture was stirred at room temperature for 1.5 hours. After thesolvent was removed in vacuo, purification was carried out by a silicagel column chromatography (silica gel 30 g, chloroform methanol=200:0-7)to afford 1.36 g of an amine derivative.

The amine derivative thus obtained was dissolved in methanol (40 ml), 5%palladium carbon (0.28 g) was added and the mixture was stirred underhydrogen atmosphere for 10 hours. The palladium carbon was filtered off,the methanol was removed in vacuo and then the residue was purified by asilica gel column chromatography (silica gel 30 g,chloroform:methanol=200:0-20) and solidified with a solvent system ofchloroform—diethyl ether to afford 0.87 g of a deprotected derivative.

The deprotected derivative thus obtained (0.60 g) was dissolved in THF(100 ml) and N-methylmorpholine (0.10 ml) and HOBt·monohydrate (0.28 g)were added to form Solution A. To a mixed solvent of THF (200 ml) andDMF (100 ml) were added in turn cesium chloride (0.76 g), potassiumchloride (0.30 g) and WSCI (0.78 g) to form Solution B. Solution A wasadded dropwise to Solution B over 20 minutes while stirring at roomtemperature and then the mixture was further stirred at room temperaturefor 10 days. The reaction solution was diluted with ethyl acetate (150ml), washed in turn with water, 5% aqueous sodium hydrogen-carbonate,water, 10% aqueous citric acid and water, and then dried over anhydroussodium sulfate. After the solvent was removed in vacuo, the residue waspurified by a silica gel column chromatography (silica gel 20 g,chloroform methanol=200:0-6) and further purified again by a silica gelcolumn chromatography (silica gel 20 g, chloroform ethyl acetate=100:10-70) and then solidified using diethyl ether and hexane to afford 0.40g of the cyclic depsipeptide (9) of the invention.

(Data for the Cyclic Depsipeptide (9))

¹H-NMR (CDCl₃) δ ppm: 7.50 (1H, d, J=7.8 Hz), 7.40 (1H, br s), 7.32 (1H,br s), 7.11-7.28 (4H, m), 7.16 (2H, d, J=8.3 Hz), 7.14 (2H, d, J=7.8Hz), 6.95 (1H, br s), 6.83 (4H, d, J=7.8 Hz), 6.15 (1H, d, J=7.8 Hz),5.06-5.13 (1H, m), 4.65-4.73 (1H, m), 4.26-4.37 (3H, m), 4.11-4.26 (2H,m), 4.06 (1H, t, J=6.6 Hz), 3.78 (6H, s), 2.78-2.92 (2H, m), 2.19-2.48(5H, m), 2.03-2.12 (2H, m), 1.77-1.91 (1H, m), 1.35-1.76 (1H, m), 1.43(9H, s), 1.07-1.34 (20H, m), 0.81-0.98 (33H, m)

SYNTHESIS EXAMPLE 52 (4)

In the same manner as described in Synthesis Example 12, 0.16 g of thecyclic depsipeptide (10) of the invention was obtained from theintermediate compound (0.30 g) obtained in Synthesis Example 52 (3).

(Data for the Cyclic Depsipeptide (10))

¹H-NMR (DMSO-d₆) δ ppm: 12.28 (1H, s), 8.72 (1H, d, J=5.4 Hz), 8.27 (1H,d, J=9.8 Hz), 8.16 (1H, d, J=7.8 Hz), 8.07 (1H, d, J=8.3 Hz), 7.92 (1H,d, J=5.9 Hz), 7.74 (1H, d, J=6.8 Hz), 7.32 (1H, d, J=9.3 Hz), 7.19 (1H,s), 6.64 (1H, s), 5.19 (1H, qui., J=6.3 Hz), 4.61-4.69 (1H, m),4.20-4.38 (3H, m), 4.06-4.16 (3H, m), 2.90-2.98 (1H, m), 2.60-2.70 (1H,m), 1.90-2.40 (5H, m), 1.60-1.80 (3H, m), 1.00-1.60 (31H, m), 0.70-0.95(33H, m)

SYNTHESIS EXAMPLE 53

To a solution of the intermediate compound (32) (2.50 g), which had beenprepared by deprotecting in the same manner as described in SynthesisExample 57 the intermediate compound obtained, starting from the benzyl3-hydroxymyristate of Synthesis Example 1, in the same manner asdescribed in Synthesis Examples 50 and 51, Fmoc-L-valine (1.16 g) andHOBt.monohydrate (0.50 g) in dichloromethane (100 ml) was added underice-cooling WSCI (0.98 g), the mixture was stirred under ice-cooling forone hour and then allowed to gradually rise up to room temperature andstirred for 3 hours. The reaction solution was washed with a saturatedaqueous solution of sodium chloride and dried over anhydrous sodiumsulfate. After the solvent was removed in vacuo, the residue wasdissolved in DMF (50 ml), diethylamine (3 ml) was added and the mixturewas stirred at room temperature for one hour. After the solvent wasremoved in vacuo, purification was carried out by a silica gel columnchromatography (silica gel 75 g, chloroform—chloroform : methanol=50:1)to afford 2.86 g of the intermediate compound (35) (SEQ ID NO 1).

(NMR Data for the Intermediate Compound (35))

¹H-NMR (δ ppm, CDCl₃) 8.26 (1H, d, J=8.3 Hz), 6.97 (1H, d, J=7.8 Hz),6.97 (1H, d, J=7.3 Hz), 7.32-7.38 (5H, m), 5.24-5.29 (1H, m), 5.11 (2H,s), 4.72-4.77 (1H, m), 4.46-4.51 (1H, m), 4.36-4.41 (1H, m), 3.27 (1H,d, J=3.9 Hz), 2.55-2.71 (4H, m), 2.24-2.32 (1H, m), 1.44 (9H, s),1.03-1.91 (28H, m), 0.81-1.00 (21H, m)

SYNTHESIS EXAMPLE 54

To a solution of the intermediate compound (35) (2.86 g) obtained inSynthesis Example 53, Fmoc-L-leucine (1.20 g) and HOBt.monohydrate (0.50g) in dichloromethane (100 ml) was added under ice-cooling WSCI (0.98g), the mixture was stirred under ice-cooling for one hour and thenallowed to gradually rise up to room temperature and stirred overnight.The reaction solution was washed with a saturated aqueous solution ofsodium chloride and dried over anhydrous sodium sulfate. After thesolvent was removed in vacuo, the residue was dissolved in DMF (50 ml),diethylamine (3 ml) was added and the mixture was stirred at roomtemperature for 1.5 hours. After the solvent was removed in vacuo,purification was carried out by a silica gel column chromatography(silica gel 100 g, chloroform—chloroform: methanol=40:1 to 20:1) toafford 2.99 g of the intermediate compound (36) (SEQ ID NO 2).

(NMR Data for the Intermediate Compound (36))

¹H-NMR (6 ppm, CDCl₃) 7.98-8.02 (1H, m), 7.53-7.56 (1H, m), 7.22-7.25(1H, m), 6.74-6.77 (1H, m), 7.31-7.36 (5H, m), 5.23-5.26 (1H, m), 5.10(2H, s), 4.61-4.67 (1H, m), 5 4.41-4.49 (2H, m), 4.07-4.11 (1H, m),3.48-3.51 (1H, m), 1.59-2.78 (16H, m), 1.42 (9H, s), 1.24 (bs, 20H),1.03-1.91 (23H, m), 0.85-1.01 (27H, m)

SYNTHESIS EXAMPLE 55

To a solution of the intermediate compound (36) (2.99 g) obtained inSynthesis Example 54, Fmoc-L-leucine (1.12 g) and HOBt.monohydrate (0.47g) in dichloromethane (50 ml) was added under ice-cooling WSCI (0.91 g),the mixture was stirred under ice-cooling for one hour and then allowedto rise up to room temperature and stirred overnight. The reactionsolution was washed with a saturated aqueous solution of sodium chlorideand dried over anhydrous sodium sulfate. After the solvent was removedin vacuo, the residue was dissolved in DMF (30 ml), diethylamine (3 ml)was added and the mixture was stirred at room temperature for 2 hours.After the solvent was removed in vacuo, purification was carried out bya silica gel column chromatography (silica gel 75 g, chloroformmethanol=20:0-1) to afford 3.46 g of the intermediate compound (37) (SEQID NO 3). To a solution of this compound, N-α-Fmoc-N-γ-Mbh-L-glutamine(1.88 g) and HOBt.monohydrate (0.47 g) in DMF (25 ml) was added underice-cooling WSCI (0.91 g), the mixture was stirred under ice-cooling forone hour and then allowed to rise up to room temperature and stirredovernight. The reaction solution was concentrated and the residue wasdissolved in DMF (100 ml), diethylamine (5 ml) was added and the mixturewas stirred at room temperature for 2 hours. After the solvent wasremoved in vacuo, purification was carried out by a silica gel columnchromatography (silica gel 150 g, chloroform: methanol=30:0-1) to afford2.55 g of the intermediate compound (38) (SEQ ID NO 4). This compoundwas dissolved in a mixed solvent of methanol (50 ml) and DMF (25 ml),10% palladium carbon (0.33 g) was added and the mixture was stirredunder hydrogen atmosphere for one hour. The palladium carbon wasfiltered off and the solvent was removed in vacuo to afford 2.52 g ofthe intermediate compound (39). This intermediate compound (39) (SEQ IDNO 5) (2.08 g) was dissolved in a mixed solvent of DMF (390 ml) and THF(1170 ml), and cesium chloride (2.82 g), potassium chloride (1.11 g),N-methylmorpholine (0.33 g), HOBt.monohydrate (0.90 g) and WSCI (2.88 g)were added in turn and the mixture was stirred at room temperature for 5days. The reaction solution was concentrated, the residue was dissolvedin ethyl acetate (400 ml), washed with water (200 ml), and then driedover anhydrous sodium sulfate. After the solvent was removed in vacuo,the residue was purified by a silica gel column chromatography (silicagel 75 g, chloroform: methanol=100:0-1) to afford 1.24 g of the cyclicdepsipeptide (11) of the invention (SEQ ID NO 6). A solution of theproduct in TFA (5 ml) was stirred at room temperature for 2 hours. Afterthe solvent was removed in vacuo, the residue was neutralized with 5%aqueous sodium hydrogencarbonate and extracted with a 10% methanolicsolution of chloroform. After the organic layer was dried over anhydroussodium sulfate, the residue was purified by a silica gel columnchromatography (silica gel 50 g, chloroform: methanol=10:0-2) to afford0.40 g of the cyclic depsipeptide (12) of the invention (hereinafterreferred to as “Compound 7”) (SEQ ID NO 7).

(NMR Data for Compound 7)

¹H-NMR (δ ppm, d-DMSO) 6.64-9.45 (9H, m), 4.93-5.12 (1H, m), 3.96-4.44(7H, m), 1.90-2.56 (8H, m), 1.10-1.77 (33H, m), 0.75-0.92 (33H, m)FAB-MS 1021 (MH⁺), 1059 (MK⁺)

SYNTHESIS EXAMPLE 56

To a solution of the intermediate compound (10) (2.46 g) obtained inSynthesis Example 27, Fmoc-D-alanine (1.89 g) and HOBt.monohydrate (0.90g) in dichloromethane (50 ml) was added under ice-cooling WSCI (1.74 g),the mixture was stirred under ice-cooling for one hour and then allowedto gradually rise up to room temperature and stirred overnight. Afterthe solvent was removed in vacuo, purification was carried out by asilica gel column chromatography (silica gel 150 g, chloroform) toafford 3.48 g of the intermediate compound (40).

(NMR Data for the Intermediate Compound (40))

¹H-NMR (δ ppm, CDCl₃) 7.76 (2H, d, J=7.8 Hz), 7.59 (2H, d, J=7.3 Hz),7.24-7.51 (9H, m), 6.52 (1H, bs), 5.26-5.30. (1H, m), 5.42 (1H, bs),5.11 (1H, d, J=12.2 Hz), 5.07 (1H, d, J=12.2 Hz), 4.21-4.50 (5H, m),2.56-2.64 (2H, m), 1.23-1.60 (26H, m), 0.88 (3H, t, J=6.8 Hz)

SYNTHESIS EXAMPLE 57

To a solution of the intermediate compound (40) (3.48 g) obtained inSynthesis Example 56 in DMF (30 ml) was added diethylamine (3 ml) andthe mixture was stirred at room temperature for one hour. After thesolvent was removed in vacuo, purification was carried out by a silicagel column chromatography (silica gel 75 g, chloroformmethanol=200:0-10) to afford 1.99 g of the intermediate compound (41).

(NMR Data for the Intermediate Compound (41))

¹H-NMR (δ ppm, CDCl₃) 7.65 (1H, bs), 7.31-7.38 (5H, m), 5.25-5.32 (1H,m), 5.13 (1H, d, J=12.2 Hz), 5.08 (1H, d, J=12.2 Hz), 4.47-4.54 (1H, m),3.48 (1H, q, J=6.8 Hz), 2.57-2.70 (2H, m), 1.24-1.60 (28H, m), 0.88 (3H,t, J=6.8 Hz)

SYNTHESIS EXAMPLE 58

To a solution of the intermediate compound (41) (1.99 g) obtained inSynthesis Example 57, Fmoc-L-aspartic acid β-t-butyl ester (1.72 g) andHOBt.monohydrate (0.62 g) in dichloromethane (200 ml) was added underice-cooling WSCI (1.20 g), the mixture was stirred under ice-cooling forone hour and then allowed to rise up to room temperature and stirredovernight. After the solvent was removed in vacuo, purification wascarried out by a silica gel column chromatography (silica gel 100 g,chloroform: methanol=50:0-1) to afford 3.42 g of the intermediatecompound (42).

(NMR Data for the Intermediate Compound (42))

¹H-NMR (δ ppm, CDCl₃) 7.76 (2H, d, J=7.3 Hz), 7.58 (2H, d, J=7.3 Hz),7.28-7.42 (9H, m), 6.97-7.01 (1H, m), 6.82-6.90 (1H, m), 5.93-5.97 (1H,m), 5.22-5.26 (1H, m), 5.10 (1H, d, J=12.2 Hz), 5.04 (1H, d, J=12.2 Hz),4.21-4.59 (6H, m), 2.55-2.68 (3H, m), 3.04 (1H, dd, J=4.4 Hz, 17.1 Hz),1.22-1.64 (35H, m), 0.88 (3H, t, J=6.3 Hz)

SYNTHESIS EXAMPLE 59

To a solution of the intermediate compound (42) (3.42 g) obtained inSynthesis Example 58 in DMF (30 ml) was added diethylamine (3 ml) andthe mixture was stirred at room temperature for one hour. After thesolvent was removed in vacuo, purification was carried out by a silicagel column chromatography (silica gel 100 g, chloroform methanol=50:0-1)to afford 2.52 g of the intermediate compound (43).

(NMR Data for the Intermediate Compound (43))

¹H-NMR (δ ppm, CDCl₃) 7.85 (1H, d, J=8.3 Hz), 7.00-7.06 (1H, m),7.31-7.35 (5H, m), 5.23-5.27 (1H, m), 5.12 (1H, d, J=12.2 Hz), 5.07 (1H,d, J=12.2 Hz), 4.45-4.55 (2H, m), 3.57-3.60 (1H, m), 2.56-2.96 (4H, m),1.24-1.66 (37H, m), 0.88 (3H, t, J=6.3 Hz)

SYNTHESIS EXAMPLE 60

To a solution of the intermediate compound (43) (1.99 g) obtained inSynthesis Example 59, Fmoc-L-alanine.monohydrate (1.27 g) andHOBt.monohydrate (0.57 g) in dichloromethane (50 ml) was added underice-cooling WSCI (1.11 g), the mixture was stirred under ice-cooling forone hour and then allowed to gradually rise up to room temperature andstirred overnight. After the solvent was removed in vacuo, purificationwas carried out by a silica gel chromatography (silica gel 100 g,chloroform: methanol=50:0-1) to afford 3.45 g of the intermediatecompound (44).

(NMR Data for the Intermediate Compound (44))

¹H-NMR (δ ppm, CDCl₃) 7.75 (2H, d, J=6.8 Hz), 7.13-7.63 (14H, m),6.88-6.91 (1H, m), 5.40-5.43 (1H, m), 5.18-5.21 (1H, m), 5.00-5.11 (2H,m), 4.72-4.78 (1H, m), 4.42-4.53 (3H, m), 4.16-4.22 (2H, m), 2.47-2.96(4H, m), 1.17-1.71 (38H, m), 0.88 (3H, t, J=6.8 Hz)

SYNTHESIS EXAMPLE 61

To a solution of the intermediate compound (44) (3.45 g) obtained inSynthesis Example 60 in DMF (30 ml) was added diethylamine (3 ml), themixture was stirred at room temperature for 2 hours. After the solventwas removed in vacuo, purification was carried out by a silica gelcolumn chromatography (silica gel 100 g, chloroform: methanol=100:0-5)to afford 2.88 g of the intermediate compound (45).

(NMR Data for the Intermediate Compound (45))

¹H-NMR (δ ppm, CDCl₃) 8.21-8.26 (1H, m), 7.13 (1H, d, J=7.8 Hz),6.88-6.95 (1H, m), 7.32-7.37 (5H, m), 5.22-5.26 (1H, m), 5.06-5.14 (2H,m), 4.68-4.74 (1H, m), 4.39-4.56 (2H, m), 3.53-3.60 (1H, m), 2.56-2.95(4H, m), 1.83 (2H, br), 1.24-1.64 (38H, m), 0.88 (3H, t, J=6.8 Hz)

SYNTHESIS EXAMPLE 62

To a solution of the intermediate compound (45) (1.50 g) obtained inSynthesis Example 61, Fmoc-D-alanine (0.68 g) and HOBt.monohydrate (0.33g) in dichloromethane (50 ml) was added under ice-cooling WSCI (0.58 g),the mixture was stirred under ice-cooling for 3 hours. After the solventwas removed in vacuo, purification was carried out by a silica gelcolumn chromatography (silica gel 100 g, chloroform: methanol=50:0-1) toafford 1.76 g of the intermediate compound (46).

(NMR Data for the Intermediate Compound (46))

¹H-NMR (δ ppm, CDCl₃) 7.76 (2H, d, J=7.3 Hz), 7.56-7.60 (2H, m),7.14-7.41 (12H, m), 6.95-7.01 (1H, m), 6.16-6.18 (1H, m), 5.24-5.27 (1H,m), 5.02-5.13 (2H, m), 4.19-4.54 (8H, m), 2.53-2.99 (4H, m), 1.14-1.66(41H, m), 0.88 (3H, t, J=6.8 Hz)

SYNTHESIS EXAMPLE 63

To a solution of the intermediate compound (46) (1.76 g) obtained inSynthesis Example 62 in DMF (30 ml) was added diethylamine (2 ml) andthe mixture was stirred at room temperature for 4 hours. After thesolvent was removed in vacuo, Fmoc-L-alanine-monohydrate (0.57 g) andHOBt.monohydrate (0.26 g) were added and dissolved in dichloromethane(30 ml). To this solution was added under ice-cooling WSCI (0.50 g) andthe mixture was stirred under ice-cooling for 3 hours. After the solventwas removed in vacuo, purification was carried out by a silica gelcolumn chromatography (silica gel 100 g, chloroform: methanol=50:0-1) toafford 1.55 g of the intermediate compound (47).

(NMR Data for the Intermediate Compound (47))

¹H-NMR (δ ppm, CDCl₃) 7.26-7.93 (18H, m), 5.40-5.48 (1H, m), 5.22-5.26(1H, m), 5.08-5.10 (2H, m), 4.08-4.77 (9H, m), 3.05-3.13 (1H, m),2.55-2.72 (3H, m), 1.21-1.67 (44H, m), 25 0.88 (3H, t, J=6.3 Hz)

SYNTHESIS EXAMPLE 64

To a solution of the intermediate compound (47) (1.55 g) obtained inSynthesis Example 63 in DMF (30 ml) was added diethylamine (2 ml) andthe mixture was stirred at room temperature for 1.5 hours. After thesolvent was removed in vacuo, purification was carried out by a silicagel column chromatography (silica gel 100 g, chloroform:methanol=50:0-5) to afford 1.10 g of the intermediate compound (48).

(NMR Data for the Intermediate Compound (48))

¹H-NMR (δ ppm, CDCl₃) 6.96-7.92 (10H, m), 5.24-5.28 (1H, m), 5.07 (1H,d, J=12.2 Hz), 5.12 (1H, d, J=12.2 Hz), 4.25-4.61 (5H, m), 3.47-3.53(1H, m), 2.83-2.95 (2H, m), 2.56-2.74 (2H, m), 1.24-2.91 (46H, m), 0.88(3H, t, J=6.8 Hz)

SYNTHESIS EXAMPLE 65

The intermediate compound (48) (1.10 g) obtained in Synthesis Example64, N-α-Fmoc-N-γ-Mbh-L-glutamine (0.77 g) and HOBt.monohydrate (0.19 g)were dissolved in a mixed solvent of dichloromethane (30 ml) and DMF (25ml), WSCI (0.36 g) was added under ice-cooling, the mixture was stirredunder ice-cooling for one hour and then allowed to rise up to roomtemperature and stirred overnight. The reaction solution wasconcentrated and the residue was dissolved in DMF (100 ml), diethylamine(2 ml) was added and the mixture was stirred at room temperature for onehour. After the solvent was removed in vacuo, purification was carriedout by a silica gel column chromatography (silica gel 100 g, chloroform:methanol=50:0-1). The purified product was dissolved in a mixed solventof methanol (100 ml) and DMF (20 ml), 10% palladium carbon (0.21 g) wasadded and the mixture was stirred under hydrogen atmosphere for onehour. The palladium carbon was filtered off and the solvent was removedin vacuo to afford 1.29 g of the intermediate compound (49).

(NMR Data for the Intermediate Compound (49))

¹H-NMR (δ ppm, CDCl₃) 8.51-8.58 (2H, m), 8.23 (1H, d, J=7.8 Hz), 8.10(1H, d, J=7.8 Hz), 7.90 (1H, bs), 7.62 (1H, bs), 7.24 (1H, d, J=7.3 Hz),7.13-7.16 (4H, m), 6.80-6.83 (4H, m), 6.09 (1H, d, J=7.8 Hz), 5.17-5.20(1H, m), 4.81-4.85 (1H, m), 4.43-4.48 (2H, m), 4.28-4.32 (1H, m),4.02-4.11 (2H, m), 3.77 (s, 6H), 3.65-3.69 (1H, m), 3.01-3.06 (1H, m),2.64-2.71 (1H, m), 2.48-2.51 (2H, m), 2.32-2.35 (2H, m), 1.95-2.06 (2H,m), 1.10-1.57 (46H, m), 0.88 (3H, t, J=6.3 Hz)

SYNTHESIS EXAMPLE 66

The intermediate compound (49) (0.12 g) obtained in Synthesis Example 65was dissolved in a mixed solvent of THF (75 ml) and DMF (25 ml), andcesium chloride (0.18 g), potassium chloride (70 mg), N-methylmorpholine(20 mg), HOBt.monohydrate (60 mg) and WSCI (0.18 g) were added in turnand the mixture was stirred at room temperature for 5 days. After thesolvent was removed in vacuo, the residue was purified by a silica gelcolumn chromatography (silica gel 30 g, chloroform: methanol=50:0-1) toafford 0.11 g of the cyclic depsipeptide (13) of the invention.

(NMR Data for the Cyclic Depsipeptide (13))

¹H-NMR (δ ppm, CDCl₃) 8.04-8.18 (2H, m), 7.77 (1H, d, J=7.8 Hz),7.55-7.60 (3H, m), 7.39-7.41 (1H, m), 7.13-7.16 (5H, m), 6.81-6.85 (4H,m), 6.11 (1H, d, J=8.3 Hz), 5.08-5.11 (1H, m), 4.77-4.81 (1H, m),4.13-4.41 (6H, m), 3.78 (s, 6H), 3.01-3.04 (1H, m), 2.63-2.67 (1H, m),2.30-2.47 (4H, m), 2.05-2.08 (2H, m), 1.25-1.67 (44H, m), 0.88 (3H, t,J=6.3 Hz)

SYNTHESIS EXAMPLE 67

The cyclic depsipeptide (13) (0.11 g) obtained in Synthesis Example 66was dissolved in TFA (3 ml) and the mixture was stirred at roomtemperature for 1.5 hours. After the solvent was removed in vacuo, tothe residue were added chloroform and water, and the aqueous layer wasneutralized with 5% aqueous sodium hydrogencarbonate. The insolublesthus precipitated out were recovered by filtration, washed with etherand dried under reduced pressure to afford 41 mg of the cyclicdepsipeptide (14) of the invention.

(NMR Data for the Cyclic Depsipeptide (14))

¹H-NMR (δ ppm, d-DMSO) 8.27 (1H, bs), 7.69-8.11 (6H, m), 7.19 (1H, bs),6.67 (1H, bs), 5.07-5.11 (1H, m), 4.17-4.34 (7H, m), 2.39-2.62 (2H, m),1.58-1.89 (4H, m), 2.09-2.12 (2H, m), 1.08-1.25 (25H, m), 0.86 (3H, t,J=6.3 Hz) FAB-MS 825 (MH⁺)

SYNTHESIS EXAMPLE 68 Fmoc-D-Leu+Val-OBzl→Fmoc-D-Leu-Val-OBzl

L-Valine benzyl ester-p-toluenesulfonate (8.35 g), Fmoc-D-leucine (7.07g) and HOBt.monohydrate (3.37 g) were dissolved in dichloromethane (80ml). To this solution was added under ice-cooling WSCI (4.22 g). Thissolution was stirred under ice-cooling for 2 hours and then allowed torise up to room temperature and stirred for 2 days. After thedichloromethane was removed in vacuo, to the residue were added ethylacetate and 10% aqueous citric acid. The separated ethyl acetate layerwas washed in turn with water, 5% aqueous sodium hydrogencarbonate andwater and then dried over anhydrous sodium sulfate. After the ethylacetate was removed in vacuo, the residue was purified by a silica gelcolumn chromatography (silica gel 80 g, chloroform: methanol=200:0-4) toafford 6.27 g of the dipeptide (4).

(NMR Data for the Dipeptide (4))

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=8.0 Hz), 7.58 (2H, d, J=7.6 Hz),7.24-7.35 (7H, m), 6.45-6.65 (1H, m), 5.17 (1H, d, J=12 Hz), 5.08 (1H,d, J=12 Hz), 5.02-5.26 (1H, m), 4.58 (1H, dd, J=4.4, 8.0 Hz), 4.40 (2H,d, J=6.8 Hz), 4.22 (1H, t, J=6.8 Hz), 4.15-4.31 (1H, m), 2.08-2.26 (1H,m), 1.53-1.79 (2H, m), 1.41-1.53 (1H, m), 0.70-0.96 (12H, m)

SYNTHESIS EXAMPLE 69 Fmoc-D-Leu-Val-OBzl→Fmoc-Leu-D-Leu-Val-OBzl

To a solution of the dipeptide (4) (5.00 g) obtained in SynthesisExample 68 in DMF (60 ml) was added diethylamine (6 ml) and the mixturewas stirred at room temperature for 2 hours. After the solvent wasremoved in vacuo, Fmoc-L-leucine (3.58 g) and HOBt.monohydrate (1.55 g)were added and dissolved in dichloromethane (30 ml). To this solutionwas added under ice-cooling WSCI (1.94 g).

This solution was stirred under ice-cooling for 2 hours and then allowedto rise up to room temperature and stirred overnight. After thedichloromethane was removed in vacuo, to the residue were added ethylacetate and 10% aqueous citric acid. The separated ethyl acetate layerwas washed in turn with water, 5% aqueous sodium hydrogencarbonate andwater and then dried over anhydrous sodium sulfate. After the ethylacetate was removed in vacuo, the residue was purified by a silica gelcolumn chromatography (silica gel 50 g, chloroform: methanol=200:0-6)and further solidified using diethyl ether and hexane to afford 5.43 gof the tripeptide (5).

(NMR Data for the Tripeptide (5))

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=7.3 Hz), 7.57 (2H, d, J=7.3 Hz),7.40 (2H, t, J=7.6 Hz), 7.20-7.36 (7H, m), 6.78 (1H, d, J=8.3 Hz), 6.36(1H, d, J=7.8 Hz), 5.13 (1H, d, J=13 Hz), 5.03 (1H, d, J=13 Hz),4.98-5.20 (1H, m), 4.41-4.53 (3H, m), 4.37 (1H, t, J=8.6 Hz), 4.08-4.26(2H, m), 2.08-2.24 (1H, m), 1.38-1.83 (6H, m), 0.74-1.03 (13H, m)

SYNTHESIS EXAMPLE 70Fmoc-Leu-D-Leu-Val-OBzl→Fmoc-Gln(Mbh)-Leu-D-Leu-Val-OBzl

To a solution of the tripeptide (5) (1.00 g) obtained in SynthesisExample 69 in DMF (12 ml) was added diethylamine (1.2 ml) and themixture was stirred at room temperature for 2 hours. After the solventwas removed in vacuo, N-α-9-Fmoc-N-γ-Mbh-L-glutamine (1.00 g) andHOBt.monohydrate (0.26 g) were added and dissolved in dichloromethane(30 ml). To this solution was added under ice-cooling WSCI (0.32 g).This solution was stirred under. ice-cooling for 2 hours and thenallowed to gradually rise up to room temperature and stirred overnight.After the dichloromethane was removed in vacuo, to the residue wereadded chloroform, ethyl acetate and 10% aqueous citric acid. Theseparated organic layer was washed in turn with water, 5% aqueous sodiumhydrogencarbonate and water and then dried over anhydrous sodiumsulfate. After the solvent was removed in vacuo, the residue wassolidified using chloroform, methanol and diethyl ether to afford 1.48 gof the tetrapeptide (8).

(NMR Data for the Tetrapeptide (8)).

¹H-NMR (CDCl₃) δ ppm: 7.75 (2H, d, J=7.3 Hz), 7.62 (2H, d, J=7.6 Hz),7.39 (2H, t, J=7.6 Hz), 7.24-7.35 (7H, m), 7.10-7.20 (4H, m), 6.78-6.88(4H, m), 6.13 (1H, d, J=8.3 Hz), 5.14 (1H, d, J=12 Hz), 5.02 (1H, d,J=14 Hz), 4.28-4.50 (5H, m), 4.16-4.24 (1H, m), 4.00-4.10 (1H, m), 3.77(3H, s), 3.75 (3H, s), 2.21-2.40 (2H, m), 2.08-2.21 (1H, m), 1.92-2.08(2H, m), 1.42-1.75 (6H, m), 0.78-0.98 (18H, m)

SYNTHESIS EXAMPLE 71Fmoc-Gln(Mbh)-Leu-D-Leu-Val-OBzl→Fmoc-Gln(Mbh)-Leu-D-Leu-Val

The tetrapeptide (8) (1.40 g) obtained in Synthesis Example 70 wasdissolved in a mixed solvent of methanol (50 ml) and DMF (40 ml), 5%palladium carbon (0.14 g) was added and the mixture was stirred underhydrogen atmosphere for 2 hours. The palladium carbon was filtered offand the solvent was removed in vacuo to afford 1.10 g of thetetrapeptide (9).

(NMR Data for the Tetrapeptide (9))

¹H-NMR (CDCl₃) δ ppm: 7.77 (2H, d, J=7.3 Hz), 7.64 (2H, d, J=8.8 Hz),7.37 (2H, t, J=7.3 Hz), 7.29 (2H, tt, J=1.1, 7.6 Hz), 7.14 (4H, dd,J=1.7, 9.3 Hz), 6.84 (4H, dd, J=2.4, 6.8 Hz), 6.09 (1H, s), 4.41-4.47(1H, m), 4.31-4.39 (3H, m), 4.25 (1H, d, J=5.9 Hz), 4.12-4.22 (1H, m),3.78-4.05 (1H, m), 3.75 (6H, s), 2.20-2.33 (2H, m), 2.09-2.20 (1H, m),1.91-2.09 (2H, m), 1.53-1.71 (6H, m), 0.82-0.99 (18H, m)

SYNTHESIS EXAMPLE 72 Fmoc-Asp(OtBu)+D-Leu-OBzl→Fmoc-Asp(OtBu)-D-Leu-OBzl

Fmoc-L-Aspartic acid β-t-butyl ester (9.05 g), D-leucine benzyl ester(4.42 g) and HOBt.monohydrate (3.37 g) were dissolved in dichloromethane(80 ml). To this solution was added under ice-cooling WSCI (4.22 g).This solution was stirred under ice-cooling for 2 hours and then allowedto rise up to room temperature and stirred overnight. After thedichloromethane was removed in vacuo, to the residue were added ethylacetate and 10% aqueous citric acid. The separated ethyl acetate layerwas washed in turn with water, 5% aqueous sodium hydrogencarbonate andwater and then dried over anhydrous sodium sulfate. After the ethylacetate was removed in vacuo, purification was carried out by a silicagel column chromatography (silica gel 80 g, chloroform: methanol200:0-4) to afford 8.36 g of the dipeptide (5).

(NMR Data for the Dipeptide (5))

¹H-NMR (CDCl₃) δ ppm: 7.77 (2H, d, J=7.3 Hz), 7.59 (2H, d, J=7.8 Hz),7.40 (2H, t, J=7.3 Hz), 7.28-7.37 (7H, m), 6.94 (1H, d, J=7.8 Hz), 5.96(1H, d, J=7.8 Hz), 5.17 (1H, d, J=12 Hz), 5.13 (1H, d, J=12 Hz),4.53-4.67 (2H, m), 4.39 (2H, d, J=6.8 Hz), 4.23 (1H, t, J=7.1 Hz), 2.89(1H, dd, J=3.2, 17 Hz), 2.62 (1H, dd, J=6.8, 17 Hz), 1.51-1.70 (3H, m),1.44 (9H, s), 0.90 (3H, d, J=2.4 Hz), 0.88 (3H, d, J=2.4 Hz)

SYNTHESIS EXAMPLE 73Fmoc-Asp(OtBu)-D-Leu-OBzl→Fmoc-Val-Asp(OtBu)-D-Leu-OBzl

To a solution of the dipeptide (5) (6.15 g) obtained in SynthesisExample 72 in DMF (100 ml) was added diethylamine (10 ml) and themixture was stirred at room temperature for 2 hours. After the solventwas removed in vacuo, Fmoc-L-valine (3.39 g) and HOBt.monohydrate (1.53g) were added and dissolved in DMF (70 ml). To this solution was addedunder ice-cooling WSCI (1.92 g). This solution was stirred underice-cooling for 2 hours and then allowed to gradually rise up to roomtemperature and stirred overnight. After the DMF was removed in vacuo,to the residue were added ethyl acetate and 10% aqueous citric acid. Theseparated ethyl acetate layer was washed in turn with water, 5% aqueoussodium hydrogencarbonate and water and then dried over anhydrous sodiumsulfate. After the ethyl acetate was removed in vacuo, the residue waspurified by a silica gel column chromatography (silica gel 50 g,chloroform: methanol=200:0-6) and further solidified using diethyl etherand hexane to afford 6.19 g of the tripeptide (6).

(NMR Data for the Tripeptide (6))

¹H-NMR (CDCl₃) δ ppm: 7.77 (2H, d, J=7.3 Hz), 7.60 (2H, d, J=8.3 Hz),7.40 (2H, dt, J=3.4, 7.3 Hz), 7.27-7.37 (7H, m), 7.22 (1H, d, J=7.8 Hz),7.08 (1H, d, J=7.3 Hz), 5.29 (1H, d, J=6.8 Hz), 5.09 (2H, s), 4.79-4.86(1H, m), 4.56-4.63 (1H, m), 4.41 (2H, d, J=7.3 Hz), 4.23 (1H, t, J=6.8Hz), 4.01 (1H, t, J=6.3 Hz), 2.90 (1H, dd, J=4.4, 17 Hz), 2.58 (1H, dd,J=6.5, 17 Hz), 2.10-2.20 (1H, m), 1.56-1.68 (3H, m), 1.42 (9H, s), 0.98(3H, d, J=6.8 Hz), 0.93 (3H, d, J=6.8 Hz), 0.85-0.91 (6H, m)

SYNTHESIS EXAMPLE 74Fmoc-Val-Asp(OtBu)-D-Leu-OBzl→Fmoc-Val-Asp(OtBu)-D-Leu

The tripeptide (6) (2.44 g) obtained In Synthesis Example 73 wasdissolved in methanol (100 ml), 5% palladium carbon (0.25 g) was addedand the mixture was stirred under hydrogen atmosphere for 2 hours. Thepalladium carbon was filtered off and the solvent was removed in vacuoto afford 2.13 g of the tripeptide (7).

SYNTHESIS EXAMPLE 75 Fmoc-Leu+D-Leu-OBzl→Fmoc-Leu-D-Leu-OBzl

To Fmoc-L-leucine (8.16 g) and D-leucine benzyl ester (4.64g) was addedHOBt.monohydrate (3.54 g) and the mixture was dissolved indichloromethane (140 ml). To this solution was added under ice-coolingWSCI (4.43 g). This solution was stirred under ice-cooling for 2 hoursand then allowed to gradually rise up to room temperature and stirredfor 4 days. After the dichloromethane was removed in vacuo, to theresidue were added ethyl acetate and 10% aqueous citric acid. Theseparated ethyl acetate layer was washed in turn with water, 5% aqueoussodium hydrogencarbonate and water and then dried over anhydrous sodiumsulfate. After the ethyl acetate was removed in vacuo, purification wascarried out by a silica gel column chromatography (silica gel 100 g,hexane: ethyl acetate=200:20-60) to afford 11.7 g of the dipeptide (6).

(NMR Data for the Dipeptide (6))

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=7.3 Hz), 7.57 (2H, d, J=7.3 Hz),7.39 (2H, t, J=7.6 Hz), 7.27-7.36 (7H, m), 6.52 (1H, d, J=7.8 Hz), 5.19(1H, d, J=7.8 Hz), 5.14 (1H, d, J=12 Hz), 5.09 (1H, d, J=12 Hz),4.60-4.68 (1H, m), 4.34-4.46 (2H, m), 4.21 (1H, t, J=7.1 Hz), 4.17-4.29(1H, m), 1.43-1.76 (6H, m), 0.93 (6H, d, J=5.4 Hz), 0.89 (6H, d, J=5.9Hz)

SYNTHESIS EXAMPLE 76 Fmoc-Leu-D-Leu-OBzl→Fmoc-Gln(Mbh)-Leu-D-Leu-OBzl

To a solution of the dipeptide (6) (4.81 g) obtained in SynthesisExample 75 in DMF (90 ml) was added diethylamine (9 ml) and the mixturewas stirred at room temperature for 2 hours. After the solvent wasremoved in vacuo, N-α-9-Fmoc-N-γ-Mbh-L-glutamine (5.14 g) andHOBt.monohydrate (1.32 g) were added and dissolved in dichloromethane(60 ml). To this solution was added under ice-cooling WSCI (1.66 g).This solution was stirred under ice-cooling for 2 hours and then allowedto gradually rise up to room temperature and stirred overnight After thedichloromethane was removed in vacuo, to the residue were added a 10%methanolic solution of chloroform and 10% aqueous citric acid. Theseparated 10% chloroform-methanol layer was washed in turn with water,5% aqueous sodium hydrogencarbonate and water and then dried overanhydrous sodium sulfate. After the 10% methanol-chloroform was removedin vacuo, the residue was solidified using chloroform and diethyl etherto afford 5.59 g of the tripeptide (8).

(NMR Data for the Tripeptide (8))

¹H-NMR (DMSO-d6) δ ppm: 8.51 (1H, d, J=8.3 Hz), 8.23 (1H, d, J=7.8 Hz),7.86 (2H, d, J=7.8 Hz), 7.78 (1H, d, J=7.8 Hz), 7.70 (2H, t, J=5.9 Hz),7.47 (1H, d, J=7.8 Hz), 7.39 (2H, t, J=7.3 Hz), 7.26-7.36 (7H, m), 7.14(4H, dd, J=1.5, 8.8 Hz), 6.83 (4H, dd, J=2.4, 8.8 Hz), 6.02 (1H, d,J=8.3 Hz), 5.06 (2H, s), 4.35-4.46 (1H, m), 4.17-4.34 (4H, m), 4.00-4.08(1H, m), 3.71 (3H, m), 3.70 (3H, s), 2.21-2.34 (2H, m), 1.90-2.01 (1H,m), 1.74-1.87 (1H, m), 1.47-1.64 (4H, m), 1.44 (2H, t, J=7.1 Hz),0.75-0.91 (12H, m)

SYNTHESIS EXAMPLE 77Fmoc-Gln(Mbh)-Leu-D-Leu-OBzl→Fmoc-Gln(Mbh)-Leu-D-Leu

The tripeptide (8) (2.73 g) obtained in Synthesis Example 76 wasdissolved in a mixed solvent of methanol (50 ml) and DMF (50 ml), 5%palladium carbon (0.27 g) was added and the mixture was stirred underhydrogen atmosphere for 2 hours. The palladium carbon was filtered offand the solvent was removed in vacuo and'solidified with diethyl etherto afford 2.46 g of the tripeptide (9).

SYNTHESIS EXAMPLE 78

In a mixed solvent of benzene (100 ml) and diethyl ether (20 ml) weredissolved 4-methylpentylaldehyde (4.80 g) and ethyl bromoacetate (10.0g). A portion (10 ml) of this solution was added to zinc powders (3.8 g)and the reaction was initiated by heating. After initiation of thereaction, the remaining solution was added dropwise while maintaining agentle refluxing. After completion of the dropwise addition, the mixturewas heated with stirring for 30 minutes. After cooling the reactionsolution, a 10% aqueous sulfuric acid solution (100 ml) was slowly addedwhile cooling. The mixture was allowed to stand and then the organiclayer was separated. The separated organic layer was washed in turn withwater, 5% aqueous sodium hydrogencarbonate and a saturated aqueoussolution of sodium chloride and then dried over anhydrous sodiumsulfate. After the solvent was removed in vacuo under reduced pressure,the residue was purified by a silica gel column chromatography (silicagel 100 g, hexane: ethyl acetate=100:20) to afford 5.0 g ofβ-hydroxyethyl ester.

(NMR Data for the p-hydroxyethyl Ester)

¹H-NMR (CDCl₃) δ ppm: 4.17 (2H, q, J=7 Hz), 3.97 (1H, m), 2.97 (1H, t,J=4 Hz), 2.53 (1H, dd, J=3, 17 Hz), 2.40 (1H, dd, J=9, 16 Hz), 1.53 (2H,m), 1.28 (3H, t, J=7 Hz), 1.30 (2H, m), 0.89 (6H, dd, J=1.7, 6.6 Hz)

SYNTHESIS EXAMPLE 79

To a solution of the β-hydroxyethyl ester (5.0 g) obtained in SynthesisExample 78 in diethyl ether (100 ml) were added dihydropyrane (8.0 g)and p-toluenesulfonic acid (0.2 g) and the mixture was stirred at roomtemperature for 2 hours. The reaction solution was washed twice with asaturated aqueous sodium hydrogencarbonate solution and once with asaturated aqueous sodium chloride, and then dried over anhydrous sodiumsulfate. The solvent was concentrated under reduced pressure to affordan oily product (7.33 g).

To a solution of the oily product (7.33 g) in methanol (50 ml) was addedunder ice-cooling an aqueous solution (20 ml) of potassium hydroxide(1.75 g). After 30 minutes, additional potassium hydroxide (2.0 g) wasadded. After 30 minutes, the methanol was removed in vacuo, and to theresidue was added water (100 ml). After washing with isopropyl ether,the aqueous layer was neutralized with dilute hydrochloric acid and thenextracted thrice with isopropyl ether. The combined isopropyl etherlayers were washed with water and a saturated aqueous solution of sodiumchloride and then dried over anhydrous magnesium sulfate. The solventwas concentrated under reduced pressure to afford 5.2 g of carboxylicacid.

(IR Data for the Carboxylic Acid)

IR (film, n): 3000 (br), 2953 (s), 2870 (m), 2700 (br), 1736 (sh), 1711(s), 1026 (s) cm⁻¹

SYNTHESIS EXAMPLE 80

A solution of the carboxylic acid (5.2 g) obtained in Synthesis Example79, benzyl bromide (7.28 g) and triethyl amine (4.3 g) in DMF (80 ml)was stirred at room temperature for 3 hours. After addition of isopropylether (100 ml) and water (100 ml), the organic layer was separated andthe aqueous layer was extracted with isopropyl ether. The extract wascombined with the organic layer and washed with water and a saturatedaqueous solution of sodium chloride and then dried over anhydrousmagnesium sulfate. After the solvent was removed in vacuo under reducedpressure, the residue was purified by a silica gel column chromatography(hexane: ethyl acetate=100:10) to afford 3.11 g of benzyl ester.

(NMR Data for the Benzyl Ester)

¹H-NMR (CDCl₃) δ ppm: 7.36 (5H, m), 5.12 (2H, 2s), 4.66 (1H, m), 4.05(1H, m), 3.85 (1H, m), 3.42 (1H, m), 2.52 and 2.72 (2H, m), 1.70 (2H,m), 1.50 (6H, m), 1.25 (2H, m), 0.86 (6H, m)

SYNTHESIS EXAMPLE 81

To a solution of the benzyl ester (3.11 g) obtained in Synthesis Example80 in methanol (70 ml) was added p-toluenesulfonic acid (0.35 g) and themixture was heated under reflux for one hour. After cooling to roomtemperature, isopropyl ether and water were added thereto. The organiclayer was separated and the aqueous layer was extracted with isopropylether. The extract was combined with the organic layer and washed withwater, a saturated aqueous sodium hydrogencarbonate solution and asaturated aqueous solution of sodium chloride and then dried overanhydrous magnesium sulfate. After the solvent was removed in vacuounder reduced pressure, the residue was purified by a silica gel columnchromatography (silica gel 70 g, hexane: ethyl acetate=100:15) to afford1.38 g of benzyl 6-methyl-3-hydroxyheptanoate.

(NMR Data for the Benzyl 6-methyl-3-hydroxyheptanoate)

¹H-NMR (CDCl₃) δ ppm: 7.36 (5H, m), 5.16 (2H, s), 4.00 (1H, br septet.,J=3 Hz), 2.57 (1H, dd, J=3, 17 Hz), 2.46 (1H, dd, J=9, 17 Hz), 1.50 (3H,m), 1.32 (1H, m), 1.21 (1H, m), 0.88 (6H, d, J=7 Hz)

SYNTHESIS EXAMPLE 82

To a solution of the benzyl 6-methyl-3-hydroxyheptanoate (0.75 g),Fmoc-L-isoleucine (1.17 g) and dimethylaminopyridine (26 mg) indichloromethane (20 ml) was added under ice-cooling DCC (0.93 g) and themixture was stirred under ice-cooling for 2 hours and then allowed togradually rise up to room temperature and stirred for 2 days. After aprecipitate was removed by filtration, the dichloromethane was removedin vacuo. To the residue were added ethyl acetate and 10% aqueous citricacid. The separated organic layer was washed in turn with water, 5%aqueous sodium hydrogencarbonate and water and then dried over anhydroussodium sulfate. After concentration, purification was carried out by asilica gel column chromatography (silica gel 30 g, hexane: ethylacetate=200:0-20) to afford 1.76 g of the intermediate compound (50).

(NMR Data for the Intermediate Compound (50))

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=7.3 Hz), 7.60 (2H, d, J=7.3 Hz),7.40 (2H, t, J=7.3 Hz), 7.27-7.37 (7H, m), 5.23-5.35 (2H, m), 5.11 (2H,s), 4.28-4.44 (3H, m), 4.23 (1H, t, J=6.8 Hz), 2.54-2.74 (2H, m),1.80-1.97 (1H, m), 1.32-1.67 (4H, m), 1.06-1.28 (3H, m), 0.76-0.98 (12H,m)

SYNTHESIS EXAMPLE 83

To a solution of the intermediate compound (50) (1.76 g) obtained inSynthesis Example 82 in DMF (30 ml) was added diethylamine (3 ml) andthe mixture was stirred at room temperature for 2 hours. After thesolvent was removed in vacuo, to a solution of the amine derivative thusobtained, Fmoc-D-leucine (1.17 g) and HOBt.monohydrate (0.51 g) indichloromethane (20 ml) was added under ice-cooling WSCI (0.63 g). Thissolution was stirred under ice-cooling for 2 hours and then allowed togradually rise up to room temperature and stirred overnight. After thedichloromethane was removed in vacuo, to the residue were added ethylacetate and 10* aqueous citric acid. The separated ethyl acetate layerwas washed in turn with water, 5% aqueous sodium hydrogencarbonate andwater and then dried over anhydrous sodium sulfate. After the ethylacetate was removed in vacuo, purification was carried out by a silicagel column chromatography (silica gel 25 g, hexane: ethylacetate=200:6-30) to afford 2.10 g of the intermediate compound (51).

(NMR Data for the Intermediate Compound (51))

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=7.8 Hz), 7.59 (2H, d, J=6.1 Hz),7.40 (2H, t, J=7.6 Hz), 7.27-7.36 (7H, m), 6.45-6.59 (1H, m), 5.21-5.33(1H, m), 50.9 (2H, s), 5.05-5.19 (1H, m), 4.33-4.56 (3H, m), 4.18-4.30(2H, m), 2.54-2.71 (2H, m), 1.79-1.96 (1H, m), 1.31-1.75 (7H, m),1.05-1.20 (3H, m), 0.78-1.00 (18H, m)

SYNTHESIS EXAMPLE 84

To a solution of the intermediate compound (51) (2.10 g) obtained inSynthesis Example 83 in DMF (30 ml) was added diethylamine (3 ml) andthe mixture was stirred at room temperature for 2 hours. After thesolvent was removed in vacuo, to a solution of the amine derivative thusobtained, Fmoc-L-aspartic acid β-t-butyl ester (1.36 g) andHOBt.monohydrate (0.51 g) in dichloromethane (20 ml) was added underice-cooling WSCI (0.63 g). This solution was stirred under ice-coolingfor 2 hours and then allowed to gradually rise up to room temperatureand stirred overnight. After the dichloromethane was removed in vacuo,to the residue were added ethyl acetate and 10% aqueous citric acid. Theseparated ethyl acetate layer was washed in turn with water, 5% aqueoussodium hydrogencarbonate and water and then dried over anhydrous sodiumsulfate. After the ethyl acetate was removed in vacuo, purification wascarried out by a silica gel column chromatography (silica gel 25 g,hexane: ethyl acetate=200:10-50) to afford 2.61 g of the intermediatecompound (52).

(NMR Data for the Intermediate Compound (52))

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=7.3 Hz), 7.53-7.62 (2H, m), 7.40(2H, t, J=7.6 Hz), 7.27-7.36 (7H, m), 6.93, 6.85 (1H, 2d, J=8.3 Hz),6.72 (1H, d, J=8.3 Hz), 5.98 (1H, d, J=8.3 Hz), 5.20-5.29 (1H, m),5.01-5.12 (2H, m), 4.34-4.61 (5H, m), 4.22 (1H, t, J=6.8 Hz), 2.90-3.00(1H, m), 2.51-2.74 (3H, m), 1.74-1.96 (2H, m), 1.34-1.68 (6H, m), 1.44(9H, 2s), 1.07-1.21 (3H, m), 0.78-0.96 (18H, m)

SYNTHESIS EXAMPLE 85

To a solution of the intermediate compound (52) (0.99 g) obtained inSynthesis Example 84 in DMF (20 ml) was added diethylamine (2 ml) andthe mixture was stirred at room temperature for 2 hours. After thesolvent was removed in vacuo, to a solution of the amine derivative thusobtained, the tetrapeptide (9) obtained in Synthesis Example 71 (1.10 g)and HOBt.monohydrate (0.18 g) in a mixed solvent of DMF (20 ml) anddichloromethane (10 ml) was added under ice-cooling WSCI (0.23 g). Thissolution was stirred under ice-cooling for 2 hours and then stirred atroom temperature overnight. After the solvent was removed in vacuo, tothe residue were added chloroform and 10% aqueous citric acid. Theseparated ethyl acetate layer was washed in turn with water, 5% aqueoussodium hydrogencarbonate and water and then dried over anhydrous sodiumsulfate. After the ethyl acetate was removed in vacuo, purification wascarried out by a silica gel column chromatography (silica gel 50 g,chloroform: methanol 200:0-4) to afford 1.76 g of the intermediatecompound (53).

(NMR Data for the Intermediate Compound (53))

¹H-NMR (CD₃OD) δ ppm: 7.75 (2H, d, J=7.3 Hz), 7.54-7.62 (2H, m), 7.37(2H, t, J=7.6 Hz), 7.24-7.34 (7H, m), 7.15 (4H, d, J=8.3 Hz), 6.84 (4H,d, J=8.3 Hz), 6.12 (1H, s), 5.16-5.27 (1H, m), 5.02-5.14 (2H, m), 4.84(1H, br s), 4.40-4.50 (2H, m), 4.12-4.50 (7H, m), 4.03 (1H, d, J=6.3Hz), 3.76 (6H, s), 2.91-2.98 (1H, m), 2.54-2.78 (3H, m), 2.39 (2H, t,J=7.6 Hz), 1.95-2.17 (3H, m), 1.33-1.81 (13H, m), 1.42 (9H, s),1.08-1.25 (3H, m), 0.76-1.03 (36H, m)

SYNTHESIS EXAMPLE 86

To a solution of the intermediate compound (53) (1.75 g) obtained inSynthesis Example 85 in DMF (30 ml) was added diethylamine (3 ml) andthe mixture was stirred at room temperature for 2 hours. After thesolvent was removed in vacuo, the amine derivative thus obtained wasdissolved in methanol (50 ml), 5% palladium carbon (0.15 g) was addedand the mixture was stirred under hydrogen atmosphere for 3 hours. Afterthe palladium carbon was filtered off and the solvent was removed invacuo, purification was carried out by a silica gel columnchromatography (silica gel 30 g, chloroform: methanol=200:0-25) toafford 1.18 g of the intermediate compound (54).

(NMR Data for the Intermediate Compound (54))

¹H-NMR (CD₃OD) δ ppm: 7.13 (4H, d, J=8.8 Hz), 6.81-6.88 (4H, m), 6.07(1H, s), 5.16-5.29 (1H, m), 4.70-4.84 (2H, m), 4.22-4.51 (3H, m),4.00-4.12 (1H, m), 3.82-3.88 (1H, m), 3.76 (6H, s), 2.89-3.04 (1H, m),2.70-2.80 (1H, m), 2.28-2.49 (4H, m), 2.05-2.19 (2H, m), 1.37-1.96 (15H,m), 1.44 (9H, s), 1.15-1.30 (3H, m), 0.79-1.02 (36H, m)

SYNTHESIS EXAMPLE 87

To a solution of the intermediate compound (54) (0.58 g) obtained inSynthesis Example 86 in THF (110 ml) were added N-methylmorpholine (0.10ml) and HOBt.monohydrate (0.29 g) to form Solution A. To a mixed solventof THF (220 ml) and DMF (110 ml) were added in turn cesium chloride(0.79 g), potassium chloride (0.31 g) and WSCI (0.81 g) to form SolutionB. Solution A was added dropwise to Solution. B at room temperature over20 minutes while stirring and further the mixture was stirred at roomtemperature for 7 days. The reaction solution was diluted with ethylacetate (100 ml) and the mixture was washed in turn with water, 5%aqueous sodium hydrogencarbonate, water, 10% aqueous citric acid andwater and then dried over anhydrous sodium sulfate. After the solventwas removed in vacuo, the residue was purified by a silica gel columnchromatography (silica gel 30 g, chloroform: methanol=200:0-6) andfurthermore solidified with diethyl ether-hexane system to afford 0.36 gof the cyclic depsipeptide (15) of the invention.

(NMR Data for the Cyclic Depsipeptide (15))

¹H-NMR (CD3OD) δ ppm: 7.10-7.17 (4H, m), 6.82-6.88 (4H, m), 6.03-6.09(1H, m), 5.15-5.23 (1H, m), 4.69-4.83 (1H, m), 4.22-4.51 (5H, m),4.04-4.11 (1H, m), 3.77, 3.76 (6H, 2s), 2.64-2.91 (2H, m), 2.51-2.59(1H, m), 2.15-2.45 (3H, m), 1.82-2.02 (3H, m), 1.36-1.78 (13H, m), 1.44(9H, s), 1.13-1.33 (4H, m), 0.76-1.02 (36H, m)

SYNTHESIS EXAMPLE 88

A solution of the cyclic depsipeptide (15) (0.35 g) obtained inSynthesis Example 87 in TFA (4 ml) was stirred at room temperature for 2hours. After the solvent was removed in vacuo, the residue wasneutralized with 5% aqueous sodium hydrogencarbonate and extracted witha 10% methanolic solution of chloroform. The organic layer was driedover anhydrous sodium sulfate and concentrated, and then purificationwas carried out by a silica gel column chromatography (silica gel 20 g,chloroform: methanol=100:0-50) to afford 0.21 g of the cyclicdepsipeptide (16) of the invention.

(NMR Data for the Cyclic Depsipeptide (16))

¹H-NMR (CD₃OD) δ ppm: 5.07-5.32 (1H, m), 4.28-4.81 (6H, m), 4.05-4.21(1H, m), 2.42-2.94 (4H, m), 2.11-2.32 (3H, m), 1.39-2.08 (1SH, m),1.13-1.36 (4H, m), 0.80-1.11 (36H, m)

SYNTHESIS EXAMPLE 89

To a solution of 3-hydroxyoctanoic acid (1.90 g) and triethylamine (1.65ml) in DMF (20 ml) was added benzyl bromide (1.41 ml) and the mixturewas stirred at room temperature for 3 days. After the solvent wasremoved in vacuo, ethyl acetate and water were added to the residue. Theseparated ethyl acetate layer was washed twice with water and then driedover anhydrous sodium sulfate. After concentration, the residue waspurified by a silica gel column chromatography (silica gel 15 g,chloroform: methanol=100:0-8) to afford 1.71 g of benzyl3-hydroxyoctanoate.

(NMR Data for the Benzyl 3-hydroxyoctanoate)

¹H-NMR (CDCl₃) δ ppm: 7.31-7.41 (5H, m), 5.16 (2H, s), 3.98-4.06 (1H,m), 2.85 (1H, d, J=3.4 Hz), 2.57 (1H, dd, J=3.2, 17 Hz), 2.46 (1H, dd,J=8.8, 17 Hz), 1.22-1.60 (8H, m), 0.88 (3H, t, J=6.8 Hz)

SYNTHESIS EXAMPLE 90

To a solution of the benzyl 3-hydroxyoctanoate (0.75 g),Fmoc-L-isoleucine (1.17 g) and dimethylamino-pyridine (26 mg) indichloromethane (20 ml) was added under ice-cooling DCC (0.93 g) and themixture was stirred under ice-cooling for 2 hours and then allowed togradually rise up to room temperature and stirred for 3 days. After aprecipitate was removed by filtration, the dichloromethane was removedin vacuo. To the residue were added ethyl acetate and 10% aqueous citricacid. The separated organic layer was washed in turn with water, 5%aqueous sodium hydrogencarbonate and water and then dried over anhydroussodium sulfate. After concentration, the residue was purified by asilica gel column chromatography (silica gel 30 g, hexane: ethylacetate=200:5-30) to afford 1.75 g of the intermediate compound (55).

(NMR Data for the Intermediate Compound (55))

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=7.3 Hz), 7.60 (2H, d, J=7.3 Hz),7.40 (2H, t, J=7.3 Hz), 7.27-7.37 (7H, m), 5.22-5.36 (2H, m), 5.11 (2H,s), 4.34-4.44 (2H, m), 4.34-4.44 (2H, m), 4.31 (1H, dd, J=4.6, 8.3 Hz),4.23 (1H, t, J=7.1 Hz), 2.54-2.74 (2H, m), 1.79-1.96 (1H, m), 1.51-1.71(2H, m), 1.05-1.46 (8H, m), 0.76-0.97 (9H, m)

SYNTHESIS EXAMPLE 91

To a solution of the intermediate compound (55) (1.75 g) obtained inSynthesis Example 90 in DMF (30 ml) was added diethylamine (3 ml) andthe mixture was stirred at room temperature for 2 hours. After thesolvent was removed in vacuo, to a solution of the amine derivative thusobtained, Fmoc-D-leucine (1.17 g) and HOBt.monohydrate (0.51 g) indichloromethane (20 ml) was added under ice-cooling WSCI (0.63 g). Thissolution was stirred under ice-cooling for 2 hours and then allowed togradually rise up to room temperature and stirred for 2 days. After thedichloromethane was removed in vacuo, to the residue were added ethylacetate and 10% aqueous citric acid. The separated ethyl acetate layerwas washed in turn with water, 5% aqueous sodium hydrogencarbonate andwater and then dried over anhydrous sodium sulfate. After the ethylacetate was removed in vacuo, purification was carried out by a silicagel column chromatography (silica gel 25 g, hexane: ethylacetate=200:5-35) to afford 2.10 g of the intermediate compound (56).

(NMR Data for the Intermediate Compound (56))

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=7.3 Hz), 7.57-7.60 (2H, m), 7.40(2H, t, J=7.3 Hz), 7.27-7.36 (7H, m), 6.45-6.59 (1H, m), 5.22-5.33 (1H,m), 5.09 (2H, s), 5.04-5.18 (1H, m), 4.33-4.56 (3H, m), 4.18-4.30 (2H,m), 5 2.53-2.70 (2H, m), 1.79-1.95 (1H, m), 1.45-1.75 (5H, m), 1.04-1.44(8H, m), 0.75-0.99 (15H, m)

SYNTHESIS EXAMPLE 92

To a solution of the intermediate compound (56) (2.10 g) obtained inSynthesis Example 91 in DMF (30 ml) was added diethylamine (3 ml) andthe mixture was stirred at room temperature for 2 hours. After thesolvent was removed in vacuo, to a solution of the amine derivative thusobtained, Fmoc-L-aspartic acid β-t-butyl ester (1.36 g) andHOBt.monohydrate (0.51 g) in dichloromethane (20 ml) was added underice-cooling WSCI (0.63 g). This solution was stirred under ice-coolingfor 2 hours and then allowed to gradually rise up to room temperatureand stirred for 3 days. After the dichloromethane was removed in vacuo,to the residue were added ethyl acetate and 10% aqueous citric acid. Theseparated ethyl acetate layer was washed in turn with water, 5% aqueoussodium hydrogencarbonate and water and then dried over anhydrous sodiumsulfate. After the ethyl acetate was removed in vacuo, purification wascarried out by a silica gel column chromatography (silica gel 25 g,hexane: ethyl acetate 200:10-60) to afford 1.94 g of the intermediatecompound (57).

(NMR Data for the Intermediate Compound (57))

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=7.3 Hz), 7.58 (2H, d, J=7.8 Hz),7.40 (2H, t, J=7.3 Hz), 7.27-7.37 (7H, m), 6.85-7.00 (1H, m), 6.75 (1H,d, J=8.3 Hz), 5.95-6.06 (1H, m), 5.21-5.31 (1H, m), 5.01-5.13 (2H, m),4.34-4.62 (5H, m), 4.22 (1H, t, J=6.8 Hz), 2.87-3.01 (1H, m), 2.50-2.75(3H, m), 1.74-1.97 (2H, m), 1.34-1.70 (4H, m), 1.44 (9H, 2s), 1.09-1.33(8H, m), 0.77-0.96 (15H, m)

SYNTHESIS EXAMPLE 93

To a solution of the intermediate compound (57) (1.30 g) obtained inSynthesis Example 92 in DMF (14 ml) was added diethylamine (1.4 ml) andthe mixture was stirred at room temperature for 3 hours. After thesolvent was removed in vacuo, to a solution of the amine derivative thusobtained, the tetrapeptide (9) (1.37 g) obtained in Synthesis Example 71and HOBt.monohydrate (0.23 g) in a mixed solvent of DMF (27 ml) anddichloromethane (16 ml) was added under ice-cooling WSCI (0.29 g). Thissolution was stirred under ice-cooling for 2 hours and then allowed togradually rise up to room temperature and stirred for 2 days. After thesolvent was removed in vacuo, to the residue were added chloroform and10% aqueous citric acid. The separated ethyl acetate layer was washed inturn with water, 5% aqueous sodium hydrogencarbonate and water and thendried over anhydrous sodium sulfate. After the ethyl acetate was removedin vacuo, purification was carried out by a silica gel columnchromatography (silica gel 50 g, chloroform: methanol=200:0-6) to afford1.61 g of the intermediate compound (58).

(NMR Data for the Intermediate Compound (58))

¹H-NMR (CD₃OD) δ ppm: 7.68-7.80 (2H, m), 7.53-7.63 (2H, m), 7.18-7.43(9H, m), 7.13 (4H, d, J=8.3 Hz), 6.82 (4H, d, J=8.3 Hz), 6.09 (1H, s),5.13-5.29 (1H, m), 4.98-5.13 (2H, m), 4.10-4.60 (9H, m), 3.98-4.06 (1H,m), 3.74 (6H, s), 2.52-3.00 (4H, m), 2.31-2.50 (2H, m), 1.96-2.20 (4H,m), 1.47-1.75 (1OH, m), 1.41 (9H, s), 1.08-1.45 (9H, m), 0.70-1.00 (33H,m)

SYNTHESIS EXAMPLE 94

To a solution of the intermediate compound (58) (1.61 g) obtained inSynthesis Example 93 in DMF (20 ml) was added diethylamine (2 ml) andthe mixture was stirred at room temperature for 3 hours. After thesolvent was removed in vacuo, purification was carried out by a silicagel column chromatography (silica gel 20 g, chloroform:methanol=100:0-3) to afford 1.00 g of the intermediate compound (59).

(NMR Data for the Intermediate Compound (59))

¹H-NMR (CD₃OD) δ ppm: 7.27-7.38 (5H, m), 7.13 (4H, d, J=7.8 Hz), 6.84(4H, d, J=8.8 Hz), 6.07 (1H, s), 5.21-5.29 (1H, m), 5.07-5.12 (2H, m),4.71-4.78 (1H, m), 4.41-4.48 (2H, m), 4.29-4.38 (1H, m), 4.21-4.27 (1H,m), 3.97-4.01 (1H, m), 3.76 (6H, s), 3.37 (1H, t, J=7.1 Hz), 2.88-2.98(1H, m), 2.62-2.79 (3H, m), 2.35 (2H, t, J=7.8 Hz), 2.08-2.17 (1H, m),1.81-1.95 (2H, m), 1.50-1.80 (11H, m), 1.43 (9H, s), 1.12-1.48 (9H, m),0.81-1.02 (33H, m)

SYNTHESIS EXAMPLE 95

To a solution of the intermediate compound (59) (1.00 g) obtained inSynthesis Example 94 in methanol (35 ml) was added 5% palladium carbon(0.1 g) and the mixture was stirred under hydrogen atmosphere for 4hours. The palladium carbon was filtered off and the methanol wasremoved in vacuo to afford an intermediate compound. The intermediatecompound was dissolved in THF (170 ml) and N-3-methylmorpholine (0.17ml) and HOBt.monohydrate (0.46 g) were added to form Solution A. To amixed solvent of THF (340 ml) and DMF (170 ml) were added in turn cesiumchloride (1.27 g), potassium chloride (0.51 g) and WSCI (1.30 g) to formSolution B. Solution A was added dropwise to Solution B at roomtemperature over 20 minutes while stirring and the mixture was furtherstirred at room temperature for 7 days. The reaction solution wasdiluted with ethyl acetate (200 ml) and the mixture was washed in turnwith water, 5% aqueous sodium hydrogen-carbonate, water, 10% aqueouscitric acid and water and then dried over anhydrous sodium sulfate.After the solvent was removed in vacuo, the residue was purified by asilica gel column chromatography (silica gel 30 g, chloroform:methanol=200:0-6) and furthermore solidified with diethyl ether andhexane to afford 0.64 g of the cyclic depsipeptide (17) of theinvention.

(NMR Data for the Cyclic Depsipeptide (17))

¹H-NMR (CD₃OD) δ ppm: 7.10-7.16 (4H, m), 6.82-6.88 (4H, m), 6.04-6.09(1H, m), 5.09-5.25 (1H, m), 4.70-4.77 (1H, m), 4.23-4.51 (5H, m),4.03-4.13 (1H, m), 3.77 (3H, s), 3.76 (3H, s), 2.64-2.91 (2H, m),2.51-2.58 (1H, m), 2.15-2.43 (3H, m), 1.82-2.03 (3H, m), 1.51-1.78 (11H,m), 1.44 (9H, s), 1.13-1.49 (9H, m), 0.77-1.04 (33H, m)

SYNTHESIS EXAMPLE 96

A solution of the cyclic depsipeptide (17) (0.64 g) obtained inSynthesis Example 95 in TFA (8 ml) was stirred at room temperature for 3hours. After the solvent was removed in vacuo, the residue wasneutralized with 5% aqueous sodium hydrogencarbonate and extracted witha 10% methanolic solution of chloroform. The organic layer was driedover anhydrous sodium sulfate and concentrated, and then purified by asilica gel column chromatography (silica gel 20 g, chloroform:methanol=100:0-50) and furthermore solidified using diethyl ether andhexane to afford 0.36 g of the cyclic depsipeptide (18) of theinvention.

(NMR Data for the Cyclic Depsipeptide (18))

¹H-NMR (CD₃OD) δ ppm: 5.13-5.33 (1H, m), 4.22-4.82 (6H, m), 4.06-4.22(1H, m), 2.39-2.93 (4H, m), 1.50-2.34 (16H, m), 1.14-1.50 (9H, m),0.79-1.09 (33H, m)

SYNTHESIS EXAMPLE 97

To a solution of 3-hydroxyhexadecanoic acid (1.90 g) and triethylamine(0.97 ml) in DMF (20 ml) was added benzyl bromide (0.83 ml) and themixture was stirred at room temperature for 3 days. After the solventwas removed in vacuo, ethyl acetate and water were added to the residue.The separated ethyl acetate layer was washed twice with water and thendried over anhydrous sodium sulfate. After concentration, the residuewas purified by a silica gel column chromatography (silica gel 15 g,chloroform: methanol=100:0-8) to afford 1.22 g of benzyl3-hydroxyhexadecanoate.

(NMR Data for the Benzyl 3-hydroxyhexadecanoate)

¹H-NMR (CDCl₃) δ ppm: 7.31-7.40 (5H, m), 5.16 (2H, s), 4.02 (1H, br s),2.84 (1H, br s), 2.56 (1H, dd, J=3.4, 17 Hz), 2.46 (1H, dd, J=8.8, 17Hz), 1.47-1.61 (2H, m), 1.37-1.47 (2H, m), 1.20-1.37 (20H, m), 0.88 (3H,t, J=6.8 Hz)

SYNTHESIS EXAMPLE 98

To a solution of the benzyl 3-hydroxyhexadecanoate (1.04 g),Fmoc-L-isoleucine (1.17 g) and dimethylamino-pyridine (26 mg) indichloromethane (20 ml) was added under ice-cooling DCC (0.93 g) and themixture was stirred under ice-cooling for 2 hours and then allowed togradually rise up to room temperature and stirred for 2 days. After aprecipitate was removed by filtration, the dichloromethane was removedin vacuo. To the residue were added ethyl acetate and 10% aqueous citricacid. The separated organic layer was washed in turn with water, 5%aqueous sodium hydrogencarbonate and water and then dried over anhydroussodium sulfate. After concentration, purification was carried out by asilica gel column chromatography (silica gel 30 g, hexane: ethylacetate=200:0-25) to afford 2.00 g of the intermediate compound (60).

(NMR Data for the Intermediate Compound (60))

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=7.3 Hz), 7.60 (2H, d, J=7.3 Hz),7.40 (2H, t, J=7.3 Hz), 7.27-7.37 (7H, m), 5.23-5.36 (2H, m), 5.11 (2H,s), 4.35-4.43 (2H, m), 4.31 (1H, dd, J=4.6, 9.3 Hz), 4.23 (1H, t, J=7.1Hz), 2.55-2.73 (2H, m), 1.80-1.94 (1H, m), 1.51-1.71 (2H, m), 1.04-1.46(24H, m), 0.78-0.98 (9H, m)

SYNTHESIS EXAMPLE 99

To a solution of the intermediate compound (60) (2.00 g) obtained inSynthesis Example 98 in DMF (30 ml) was added diethylamine (3 ml) andthe mixture was stirred at room temperature for 2 hours. After thesolvent was removed in vacuo, to a solution of the amine derivative thusobtained, Fmoc-D-leucine (1.12 g) and HOBt.monohydrate (0.48 g) indichloromethane (20 ml) was added under ice-cooling WSCI (0.60 g). Thissolution was stirred under ice-cooling for 2 hours and then allowed togradually rise up to room temperature and stirred for 2 days. After thedichloromethane was removed in vacuo, to the residue were added ethylacetate and 10% aqueous citric acid. The separated ethyl acetate layerwas washed in turn with water, 5% aqueous sodium hydrogencarbonate andwater and then dried over anhydrous sodium sulfate. After the ethylacetate was removed in vacuo, purification was carried out by a silicagel column chromatography (silica gel 25 g, hexane: ethylacetate=200:5-30) to afford 2.35 g of the intermediate compound (61).

(NMR Data for the Intermediate Compound (61))

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=7.8 Hz), 7.57-7.60 (2H, m), 7.39(2H, t, J=7.6 Hz), 7.27-7.36 (7H, m), 6.44-6.60 (1H, m), 5.22-5.34 (1H,m), 5.09 (2H, s), 5.04-5.18 (1H, m), 4.32-4.55 (3H, m), 4.18-4.30 (2H,m), 2.53-2.70 (2H, m), 1.79-1.96 (1H, m), 1.44-1.76 (5H, m), 1.02-1.44(24H, m), 0.78-1.00 (15H, m)

SYNTHESIS EXAMPLE 100

To a solution of the intermediate compound (61) (2.35 g) obtained inSynthesis Example 99 in DMF (30 ml) was added diethylamine (3 ml) andthe mixture was stirred at room temperature for 3 hours. After thesolvent was removed in vacuo, to a solution of the amine derivative thusobtained, Fmoc-L-aspartic acid β-t-butyl ester (1.31 g) andHOBt.monohydrate (0.49 g) in dichloromethane (20 ml) was added underice-cooling WSCI (0.61 g). This solution was stirred under ice-coolingfor 2 hours and then allowed to gradually rise up to room temperatureand stirred overnight. After the dichloromethane was removed in vacuo,to the residue were added ethyl acetate and 10% aqueous citric acid. Theseparated ethyl acetate layer was washed in turn with water, 5% aqueoussodium hydrogencarbonate and water and then dried over anhydrous sodiumsulfate. After the ethyl acetate was removed in vacuo, purification wascarried out by a silica gel column chromatography (silica gel 25 g,hexane: ethyl acetate=200:10-50) to afford 2.57 g of the intermediatecompound (62).

(NMR Data for the Intermediate Compound (62))

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=7.8 Hz), 7.58 (2H, d, J=7.3 Hz),7.39 (2H, t, J=7.3 Hz), 7.27-7.36 (7H, m), 6.84-6.93 (1H, m), 6.69-6.75(1H, m), 5.95-6.05 (1H, m), 5.20-5.30 (1H, m), 5.01-5.12 (2H, m),4.34-4.62 (5H, m), 4.22 (1H, t, J=7.1 Hz), 2.89-3.00 (1H, m), 2.50-2.75(3H, m), 1.74-1.97 (2H, m), 1.34-1.70 (4H, m), 1.44 (9H, 2s), 1.10-1.34(24H, m), 0.82-0.98 (15H, m)

SYNTHESIS EXAMPLE 101

To a solution of the intermediate compound (62) (1.34 g) obtained inSynthesis Example 100 in DMF (14 ml) was added diethylamine (1.4 ml) andthe mixture was stirred at room temperature for 3 hours. After thesolvent was removed in vacuo, to a solution of the amine derivative thusobtained, the tetrapeptide (9) (1.25 g) obtained in Synthesis Example 71and HOBt.monohydrate (0.21 g) in a mixed solvent of DMF (25 ml) anddichloromethane (15 ml) was added under ice-cooling WSCI (0.26 g). Thissolution was stirred under ice-cooling for 2 hours and then allowed togradually rise up to room temperature and stirred for 2 days. After thesolvent was removed in vacuo, to the residue were added chloroform and10% aqueous citric acid. The separated ethyl acetate layer was washed inturn with water, 5% aqueous sodium hydrogencarbonate and water and thendried over anhydrous sodium sulfate. After the ethyl acetate was removedin vacuo, purification was carried out by a silica gel columnchromatography (silica gel 50 g, chloroform: methanol=200:0-6) to afford1.28 g of the intermediate compound (63).

(NMR Data for the Intermediate Compound (63))

¹H-NMR (CD₃OD) δ ppm: 7.69-7.79 (2H, m), 7.56-7.63 (2H, m), 7.23-7.40(9H, m), 7.14 (4H, d, J=8.8 Hz), 6.83 (4H, dd, J=1.8, 8.8 Hz), 6.10 (1H,s), 5.18-5.27 (1H, m), 5.03-5.10 (2H, m), 4.21-4.61 (SH, m), 4.13-4.20(1H, m), 4.00-4.05 (1H, m), 3.75 (6H, s), 2.53-2.91 (4H, m), 2.40 (2H,t, J=7.6 Hz), 2.10-2.19 (1H, m), 1.95-2.06 (2H, m), 1.83-1.93 (1H, m),1.48-1.81 (10H, m), 1.42 (9H, s), 1.13-1.46 (25H, m), 0.79-1.00 (33H, m)

SYNTHESIS EXAMPLE 102

To a solution of the intermediate compound (63) (1.28 g) obtained inSynthesis Example 101 in DMF (15 ml) was added diethylamine (1.5 ml) andthe mixture was stirred at room temperature for 3 hours. After thesolvent was removed in vacuo, purification was carried out by a silicagel column chromatography (silica gel 20 g, chloroform:methanol=100:0-3.5) to afford 0.82 g of the intermediate compound (64).

(NMR Data for the Intermediate Compound (64))

¹H-NMR(CD₃OD) δ ppm: 7.27-7.38 (5H, m), 7.13 (4H, d, J=8.3 Hz), 6.84(4H, d, J=8.8 Hz), 6.07 (1H, s), 5.21-5.29 (1H, m), 5.08-5.12 (2H, m),4.71-4.79(1H, m), 4.41-4.49 (2H, m), 4.29-4.39 (1H, m), 4.20-4.28 (1H,m), 3.97-4.01 (1H, m), 3.76 (6H, s), 3.30-3.40 (1H, m), 2.89-3.01 (1H,m), 2.62-2.79 (3H, m), 2.36 (2H, t, J=7.8 Hz), 2.09-2.18 (1H, m),1.83-1.95 (2H, m), 1.51-1.77 (11H, m), 1.43 (9H, s), 1.12-1.47 (25H, m),0.81-1.01 (33H, m)

SYNTHESIS EXAMPLE 103

To a solution of the intermediate compound (64) (0.82 g) obtained inSynthesis Example 102 in methanol (25 ml) was added 5% palladium carbon(0.08 g) and the mixture was stirred under hydrogen atmosphere for 4hours. The palladium carbon was filtered off and the methanol wasremoved in vacuo to afford a depsipeptide. This intermediate compoundwas dissolved in THF (130 ml) and N-methyl-morpholine (0.13 ml) andHOBt.monohydrate (0.35 g) were added to form Solution A. To a mixedsolvent of THF (260 ml) and DMF (130 ml) were added in turn cesiumchloride (0.96 g), potassium chloride (0.38 g) and WSCI (0.98 g) to formSolution B. Solution A was added dropwise to Solution B at roomtemperature over 20 minutes while stirring and the mixture was furtherstirred at room temperature for 11 days. The reaction solution wasdiluted with ethyl acetate (150 ml) and the mixture was washed in turnwith water, 5% aqueous sodium hydrogen-carbonate, water, 10% aqueouscitric acid and water and then dried over anhydrous sodium sulfate.After the solvent was removed in vacuo, the residue was purified by asilica gel column chromatography (silica gel 30 g,chloroform:methanol=200:0-6) and furthermore solidified with diethylether and hexane to afford 0.60 g of the cyclic depsipeptide (19) of theinvention.

(NMR Data for the Cyclic Depsipeptide (19))

¹H-NMR (CD₃OD) δ ppm: 7.10-7.16 (4H, m), 6.82-6.89 (4H, m), 6.04-6.09(1H, m), 5.11-5.25 (1H, m), 4.69-4.76 (1H, m), 4.22-4.51 (5H, m),4.03-4.13 (1H, m), 3.77 (3H, s), 3.76 (3H, s), 2.64-2.92 (2H, m),2.51-2.58 (1H, m), 2.15-2.43 (3H, m), 1.81-2.04 (4H, m), 1.51-1.78 (10H,m), 1.44 (9H, s), 1.11-1.48 (25H, m), 0.77-1.05 (33H, m)

SYNTHESIS EXAMPLE 104

A solution of the cyclic depsipeptide (19) (0.60 g) obtained inSynthesis Example 103 in TFA (7 ml) was stirred at room temperature for3 hours. After the solvent was removed in vacuo, the residue wasneutralized with 5% aqueous sodium hydrogen-carbonate and extracted witha 10% methanolic solution of chloroform. The organic layer was driedover anhydrous sodium sulfate and concentrated, and then purified by asilica gel column chromatography (silica gel 20 g,chloroform:methanol=100:0-40) and furthermore solidified using diethylether-hexane to afford 0.33 g of the cyclic depsipeptide (20) of theinvention (hereinafter referred to as “Compound 8”).

(NMR Data for Compound 8)

¹H-NMR (CD₃OD) δ ppm: 5.12-5.33 (1H, m), 4.67-4.80 (1H, m), 4.26-4.63(5H, m), 4.04-4.20 (1H, m), 2.38-2.93 (4H, m), 1.50-2.34 (16H, m),1.17-1.50 (25H, m), 0.81-1.09 (33H, m)

SYNTHESIS EXAMPLE 105

To a 40% ethanolic solution (15 ml) of sodium cyanide (2.11 g) was addeda 40% ethanolic solution (6 ml) of (R)-(+)-1,2-epoxy-3-nonyloxypropane(2.73 g). The reaction solution was heated under reflux for 8 hours andthen the ethanol was removed in vacuo. To the residue was added undercooling 1N aqueous hydrochloric acid to adjust to pH 4 and the mixturewas then extracted with chloroform. The combined chloroform layers weredried over anhydrous sodium sulfate and the solvent was removed in vacuoto afford the intermediate compound carboxylic acid.

A solution of the carboxylic acid thus obtained, triethylamine (1.90 ml)and benzyl bromide (1.61 ml) in DMF (40 ml) was stirred at roomtemperature overnight. After the solvent was removed in vacuo, ethylacetate and water were added to the residue. The separated ethyl acetatelayer was washed twice with water and then dried over anhydrous sodiumsulfate. After the ethyl acetate was removed in vacuo, the residue waspurified by a silica gel column chromatography (silica gel 20 g,hexane:ethyl acetate=200:0-20) to afford 1.51 g of benzylnonyloxyhydroxybutanoate.

(NMR Data for Benzyl Nonyloxyhydroxybutanoate)

¹H-NMR (CDCl₃) δ ppm: 7.29-7.39 (5H, m), 5.15 (2H, s), 4.18-4.27 (1H,m), 3.36-3.49 (4H, m), 2.93 (1H, d, J=3.9 Hz), 2.58 (2H, d, J=6.3 Hz),1.55 (2H, quint., J=6.8 Hz), 1.19-1.36 (12H, m), 0.88 (3H, t, J=6.8 Hz)

SYNTHESIS EXAMPLE 106

To a solution of the ester (1.50 g) obtained in Synthesis Example 105,Fmoc-L-isoleucine (1.73 g) and dimethylaminopyridine (38 mg) indichloromethane (30 ml) was added under ice-cooling DCC (1.38 g) and themixture was stirred under ice-cooling for 2 hours and then allowed togradually rise up to room temperature and stirred for 3 days. After aprecipitate was removed by filtration, the dichloromethane was removedin vacuo. To the residue were added ethyl acetate and 10% aqueous citricacid. The separated organic layer was washed in turn with water, 5%aqueous sodium hydrogencarbonate and water and then dried over anhydroussodium sulfate. After concentration, purification was carried out by asilica gel column chromatography (silica gel 50 g, hexane:ethylacetate=200:0-30) to afford 2.41 g of the intermediate compound (65).

(NMR Data for the Intermediate Compound (65))

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=7.8 Hz), 7.60 (2H, d, J=4.9 Hz),7.40 (2H, t, J=7.3 Hz), 7.27-7.37 (7H, m), 5.39-5.48 (1H, m), 5.32 (1H,d, J=8.8 Hz), 5.12 (2H, s), 4.36-4.42 (2H, m), 4.32 (1H, dd, J=4.9, 8.8Hz), 4.23 (1H, t, J=6.8 Hz), 3.45-3.60 (2H, m), 3.31-3.45 (2H, m), 2.74(2H, d, J=6.8 Hz), 1.88 (1H, br s), 1.38-1.56 (2H, m), 1.11-1.34 (14H,m), 0.79-0.98 (9H, m)

SYNTHESIS EXAMPLE 107

To a solution of the intermediate compound (65) (2.09 g) obtained inSynthesis Example 106 in DMF (30 ml) was added diethylamine (3 ml) andthe mixture was stirred at room temperature for 3 hours. After thesolvent was removed in vacuo, to a solution of the amine derivative thusobtained, the tripeptide (6) (2.13 g) obtained in Synthesis Example 74and HOBt.monohydrate (0.52 g) in dichloromethane (25 ml) was added underice-cooling WSCI (0.66 g). This solution was stirred under ice-coolingfor 2 hours and then stirred at room temperature for 2 days. After thesolvent was removed in vacuc, to the residue were added ethyl acetateand 10% aqueous citric acid. The separated ethyl acetate layer waswashed in turn with water, 5% aqueous sodium hydrogencarbonate and waterand then dried over anhydrous sodium sulfate. After the ethyl acetatewas removed in vacuo, the residue was purified by a silica gel columnchromatography (silica gel 50 g, chloroform:) methanol 200:0-3) andfurthermore solidified using diethyl ether and hexane to afford 1.26 gof the desired intermediate compound (66).

(NMR Data for the Intermediate Compound (66))

¹H-NMR (CDCl₃) δ ppm: 7.75 (2H, d, J=7.3 Hz), 7.61 (2H, t, J=7.3 Hz),7.38 (2H, t, J=7.6 Hz), 7.23-7.36 (8H, m), 7.02 (1H, d, J=7.8 Hz), 6.91(1H, d, 8.3 Hz), 5.57 (1H, d, J=5.9 Hz), 5.33-5.40 (1H, m), 5.02-5.10(2H, m), 4.80-4.89 (1H, m), 4.35-4.57 (4H, m), 4.22 (1H, t, J=6.8 Hz),3.97 (1H, br s), 3.50 (1H, dd, J=5.4, 11 Hz), 3.44 (1H, dd, J=4.4, 11Hz), 3.28-3.39 (2H, m), 2.85 (1H, dd, J=5.9, 17 Hz), 2.75 (1H, dd,J=5.9, 18 Hz), 2.66 (2H, d, J=6.3 Hz), 2.06-2.15 (1H, m), 1.71-1.93 (4H,m), 1.55-1.67 (2H, m), 1.35-1.51 (2H, m), 1.42 (9H, s), 1.15-1.33 (12H,m), 0.82-1.01 (21H, m)

SYNTHESIS EXAMPLE 108

To a solution of the intermediate compound (66) (1.10 g) obtained inSynthesis Example 107 in DMF (10 ml) was added diethylamine (1 ml) andthe mixture was stirred at room temperature for 3 hours. After thesolvent was removed in vacuo, to a solution of the amine derivative thusobtained, the tripeptide (9) (0.94 g) obtained in Synthesis Example 77and HOBt.monohydrate (0.18 g) in DMF (10 ml) was added under ice-coolingWSCI (0.22 g). This solution was stirred under ice-cooling for 2 hoursand then allowed to gradually rise up to room temperature and stirredfor 2 days. After the solvent was removed in vacuo, to the residue wereadded chloroform and 10% aqueous citric acid. The separated chloroformlayer was washed in turn with water, 5% aqueous sodium hydrogencarbonateand water and then dried over anhydrous sodium sulfate. After thechloroform was removed in vacuo, purification was carried out by asilica gel column chromatography (silica gel 30 g, chloroform:ethylacetate=200:0-60) to afford an intermediate compound.

To a solution of the intermediate compound thus obtained in DMF (18 ml)was added diethylamine (1.8 ml) and the mixture was stirred at roomtemperature for 3 hours. After the solvent was removed in vacuo,purification was carried out by a silica gel column chromatography(silica gel 30 g, chloroform:methanol=200:0-6) to afford 0.56 g of theintermediate compound (67).

(NMR Data for the Intermediate Compound (67))

¹H-NMR (CDCl₃) δ ppm: 8.31 (1H, d, J=8.3 Hz), 7.52 (1H, d, J=9.3 Hz),7.44 (1H, d, J=4.9 Hz), 7.24-7.38 (6H, m), 7.21 (1H, d, J=5.4 Hz), 7.14(4H, dd, J=2.9, 8.8 Hz), 7.00 (1H, d, J=7.8 Hz), 6.82 (4H, dd, J=2.4,8.8 Hz), 6.67 (1H, d, J=7.8 Hz), 6.17 (1H, d, J=8.3 Hz), 5.33-5.40 (1H,m), 5.13-5.18 (1H, m), 5.10 (2H, s), 4.37-4.55 (3H, m), 3.93-4.01 (2H,m), 3.95 (1H, t, J=5.4 Hz), 3.77 (6H, s), 3.49 (2H, t, J=4.4 Hz),3.28-3.40 (3H, m), 3.09 (1H, dd, J=3.7, 16 Hz), 2.72 (2H, d, J=6.8 Hz),2.66 (1H, dd, J=10, 16 Hz), 2.42-2.52 (1H, m), 2.31-2.40 (1H, m),2.00-2.10 (1H, m), 1.89-1.98 (2H, m), 1.37 (9H, s), 1.34-1.88 (13H, m),1.12-1.33 (14H, m), 0.80-1.01 (33H, m)

SYNTHESIS EXAMPLE 109

To a solution of the intermediate compound (67) (0.46 g) obtained inSynthesis Example 108 in methanol (20 ml) was added 5% palladium carbon(0.05 g) and the mixture was stirred under hydrogen atmosphere for 3hours. The palladium carbon was filtered off, the methanol was removedin vacuo and the residue was purified by a silica gel columnchromatography (silica gel 20 g, chloroform:methanol=200:) 0-10) toafford 0.25 g of an intermediate compound. The intermediate compoundthus obtained was dissolved in THF (43 ml) and N-methylmorpholine (0.04ml) and HOBt.monohydrate (0.12 g) were added to form Solution A. To amixed solvent of THF (87 ml) and DMF (43 ml) were added in turn cesiumchloride (0.32 g), potassium chloride (0.13 g) and WSCI (0.33 g) to formSolution B. Solution A was added dropwise to Solution B at roomtemperature over one hour while stirring and the mixture was furtherstirred at room temperature for 7 days. The reaction solution wasdiluted with ethyl acetate (50 ml) and the mixture was washed in turnwith water, 5% aqueous sodium hydrogencarbonate, water, 10% aqueouscitric acid and water and then dried over anhydrous sodium sulfate.After the solvent was removed in vacuo, the residue was purified by asilica gel column chromatography (silica gel 20 g,chloroform:methanol=200:) 0-3) to afford 0.18 g of the cyclicdepsipeptide (21) of the invention.

(NMR Data for the Cyclic Depsipeptide (21))

¹H-NMR (CDCl₃) δ ppm: 7.69 (1H, d, J=8.8 Hz), 7.37 (1H, d, J=7.8 Hz),7.16 (4H, dd, J=8.8, 13 Hz), 7.07-7.13 (2H, m), 7.03 (1H, d, J=8.8 Hz),7.01 (1H, d, J=8.3 Hz), 6.90 (1H, d, 7.8 Hz), 6.86 (4H, dd, J=2.0, 8.3Hz), 6.63 (1H, br s), 6.22 (1H, d, J=8.3 Hz), 5.08-5.17 (2H, m),4.36-4.49 (3H, m), 4.19 (1H, dd, J=4.4, 7.3 Hz), 4.11 (1H, dt, J=32.9,7.6 Hz), 3.86 (1H, dd, J=4.9, 6.3 Hz), 3.79 (6H, 2s), 3.75-3.84 (2H, m),3.53 (1H, dd, J=4.2,11 Hz), 3.35-3.46 (2H, m), 3.15 (1H, dd, J=4.4, 16Hz), 2.67 (1H, dd, J=5.4, 15 Hz), 2.49 (1H, dd, J=10, 16 Hz), 2.45 (1H,dd, J=5.1, 15 Hz), 2.27-2.36 (1H, m), 2.17-2.26 (1H, m), 1.99-2.10 (1H,m), 1.47-1.99 (15H, m), 1.41 (9H, s), 1.09-1.44 (12H, m), 0.82-1.00(33H, m)

SYNTHESIS EXAMPLE 110

A solution of the cyclic depsipeptide (21) (0.12 g) obtained inSynthesis Example 109 in TFA (3 ml) was stirred at room temperature for3 hours. After the solvent was removed in vacuo, the residue wasneutralized with 5% aqueous sodium hydrogencarbonate and extracted witha 10% methanolic solution of chloroform. The organic layer was driedover anhydrous sodium sulfate and concentrated, and then purified by asilica gel column chromatography (silica gel 5 g, chloroform:methanol50:0-15) and furthermore solidified using diethyl ether-hexane to afford0.04 g of the cyclic depsipeptide (22) of the invention.

(NMR Data for the Cyclic Depsipeptide (22))

¹H-NMR (DMSO-d6) δ ppm: 9.75 (1H, br s), 9.40 (1H, br s), 8.42-8.50 (1H,m), 8.23 (1H, d, J=8.8 Hz), 7.99 (1H, d, J=8.8 Hz), 7.46-7.61 (1H, m),7.15 (2H, br s), 6.61 (1H, br s), 5.07 (1H, br s), 4.20-4.45 (5H, m),4.15 (1H, t, J=8.3 Hz), 4.06 (1H, t, J=7.3 Hz), 3.49-3.57 (2H, m),3.29-3.44 (2H, m), 2.63-2.73 (2H, m), 2.42 (1H, dd, J=7.3, 14 Hz),1.90-2.31 (5H, m), 1.73-1.87 (2H, m), 1.33-1.62 (12H, m), 1.17-1.32(13H, m), 1.06-1.16 (1H, m), 0.73-0.90 (33H, m)

SYNTHESIS EXAMPLE 111

To a solution of the intermediate compound (5) (1.30 g) obtained inSynthesis Example 6 in dimethyl-formamide (13 ml) was added diethylamine(1.3 ml) and the mixture was stirred at room temperature for 2 hours.After the solvent was removed in vacuo, N-α-9-Fmoc-N-γ-Mbh-L-glutamine(0.81 g) and 1-hydroxybenzotriazole-monohydrate (0.21 g) were added andthe mixture was dissolved in dichloromethane (30 ml). To this solutionwas added under ice-cooling1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-hydrochloride (0.26 g).This solution was stirred under ice-cooling for 2 hours and then allowedto gradually rise up to room temperature and stirred overnight. Afterthe dichloromethane was removed in vacuo, to the residue were addedchloroform and 10% aqueous citric acid. The separated chloroform layerwas washed in turn with water, 5% aqueous sodium hydrogencarbonate andwater and then dried over anhydrous sodium sulfate. After the chloroformwas removed in vacuo, the residue was purified by a silica gel columnchromatography (silica gel 25 g, chloroform:methanol=200:0-4) andfurthermore solidified using ethyl acetate and hexane to afford 1.53 gof the intermediate compound (68).

(NMR Data for the Intermediate Compound (68))

¹H-NMR (DMSO-d6) δ ppm: 8.50 (1H, d, J=9.3 Hz), 8.21 (1H, d, J=7.8 Hz),7.94 (1H, d, J=7.8 Hz), 7.85 (2H, d, J=7.3 Hz), 7.59-7.76 (4H, m), 7.48(1H, d, J=8.3 Hz), 7.39 (2H, t, J=7.6 Hz), 7.27-7.36 (7H, m), 7.14 (4H,dd, J=2.0, 8.8 Hz), 6.83 (4H, dd, J=2.7, 8.8 Hz), 6.02 (1H, d, J=8.3Hz), 5.08-5.16 (1H, m), 5.07-5.06 (2H, s), 4.57-4.65 (1H, m), 4.46-4.44(1H, m), 4.15-4.31 (5H, m), 4.04-4.12 (1H, m), 3.71 (6H, 2s), 2.58-2.71(3H, m), 2.40-2.55 (1H, m), 2.22-2.34 (2H, m), 1.90-2.00 (2H, m),1.72-1.85 (2H, m), 1.40-1.60 (5H, m), 1.32 (9H, s), 1.10-1.37 (20H, m),0.74-0.89 (21H, m)

SYNTHESIS EXAMPLE 112

To a solution of the intermediate compound (68) (1.53 g) obtained inSynthesis Example 111 in DMF (20 ml) was added diethylamine (2 ml) andthe mixture was stirred at room temperature for 2 hours. After thesolvent was removed in vacuo, to a solution of the amine derivative thusobtained in methanol (50 ml) was added 5% palladium carbon (0.15 g) andthe mixture was stirred under hydrogen atmosphere for 3 hours. Thepalladium carbon was filtered off and the methanol was removed in vacuo,the residue was purified by a silica gel column chromatography (silicagel 30 g, chloroform:methanol=200:0-30) to afford 0.77 g of theintermediate compound (69).

(NMR Data for the Intermediate Compound (69))

¹H-NMR (CD₃OD) δ ppm: 7.22 (2H, d, J=8.3 Hz), 7.14 (2H, d, J=8.8 Hz),6.88 (2H, d, J=8.8 Hz), 6.85 (2H, d, J=38.8 Hz), 6.04 (1H, s), 5.19-5.27(1H, m), 4.59-4.65 (2H, m), 4.30 (1H, d, J=4.9 Hz), 4.00-4.06 (1H, m),3.87 (1H, d, J=37.3 Hz), 3.79 (3H, s), 3.77 (3H, s), 2.87 (1H, dd,J=4.4, 17 Hz), 2.61 (1H, dd, J=8.8, 17 Hz), 2.34-2.52 (3H, m), 2.17-2.34(3H, m), 1.92-2.07 (2H, m), 1.42 (9H, s), 1.39-1.64 (5H, m), 1.20-1.35(20H, m), 0.85-1.04 (21H, m)

SYNTHESIS EXAMPLE 113

To a solution of the intermediate compound (69) (0.50 g) obtained inSynthesis Example 112 in THF (110 ml) were added N-methylmorpholine(0.10 ml) and HOBt.monohydrate (0.28 g) to form Solution A. To a mixedsolvent of THF (220 ml) and DMF (110 ml) were added in turn cesiumchloride (0.77 g), potassium chloride (0.31 g) and WSCI (0.79 g) to formSolution B. Solution A was added dropwise to Solution B at roomtemperature over 20 minutes while stirring and the mixture was furtherstirred at room temperature for 5 days. The reaction solution wasdiluted with ethyl acetate (100 ml) and the mixture was washed in turnwith water, 5% aqueous sodium hydrogencarbonate, water, 10% aqueouscitric-acid and water and then dried over anhydrous sodium sulfate.After the solvent was removed in vacuo, the residue was purified by asilica gel column chromatography (silica gel 30 g,chloroform:methanol=200:0-6) to afford 0.49 g of the cyclic depsipeptide(23) of the invention.

(NMR Data for the Cyclic Depsipeptide (23))

¹H-NMR (CD₃OD) δ ppm: 7.11-7.22 (4H, m), 6.81-6.90 (4H, m), 6.09 (1H,s), 5.17-5.30 (1H, m), 4.52-4.59 (1H, m), 4.26-4.50 (2H, m), 4.11-4.17(1H, m), 3.89-4.03 (1H, m), 3.79 (6H, 2s), 3.00-3.09 (1H, m), 2.69-2.78(1H, m), 2.29-2.57 (4H, m), 1.84-2.22 (4H, m), 1.39-1.77 (5H, m), 1.44(9H, s), 1.11-1.39 (20H, m), 0.84-1.01 (21H, m)

SYNTHESIS EXAMPLE 114

A solution of the cyclic depsipeptide (23) (0.49 g) obtained inSynthesis Example 113 in TFA (5 ml) was stirred at room temperature for2 hours. After the solvent was removed in vacuo, the residue wasneutralized with 5% aqueous sodium hydrogencarbonate and extracted witha 10% methanolic solution of chloroform. The organic layer was driedover anhydrous sodium sulfate and concentrated and then purified by asilica gel column chromatography (silica gel 20 g,chloroform:methanol=100:0-40) to afford 0.28 g of the cyclicdepsipeptide (24) of the invention.

(NMR Data for the Cyclic Depsipeptide (24))

¹H-NMR (CD₃OD) δ ppm: 5.25-5.34 (1H, m), 4.62 (1H, t, J=5.4 Hz), 4.47(1H, t, J=7.1 Hz), 4.34 (1H, dd, J=4.6, 8.8 Hz), 4.23 (1H, d, J=8.3 Hz),4.02 (1H, d, J=5.9 Hz), 2.76 (2H, d, J=5.4 Hz), 2.62 (1H, dd, J=3.4, 16Hz), 2.55 (1H, dd, J=7.3, 15 Hz), 2.38 (1H, dd, J=6.8, 8.3 Hz), 2.35(1H, d, J=5.9 Hz), 2.14-2.31 (2H, m), 1.89-2.10 (2H, m), 1.59-1.78 (4H,m), 1.47-1.56 (1H, m), 1.16-1.41 (20H, m), 0.83-1.06 (21H, m)

SYNTHESIS EXAMPLE 115

To a solution of the intermediate compound (6) (0.80 g) obtained inSynthesis Example 7 in dimethyl-formamide (8 ml) was added diethylamine(0.8 ml) and the mixture was stirred at room temperature for 4 hours.After the solvent was removed in vacuo, N-α-9-Fmoc-N-γ-Mbh-L-glutamine(0.45 g) and 1-hydroxybenzotriazole.monohydrate (0.12 g) were added andthe mixture was dissolved in dichloromethane (15 ml). To this solutionwas added under ice-cooling WSCI (0.14 g). This solution was stirredunder ice-cooling for 2 hours and then allowed to gradually rise up toroom temperature and stirred overnight. After removal of thedichloromethane, to the residue were added chloroform and 10% aqueouscitric acid. The separated chloroform layer was washed in turn withwater, 5% aqueous sodium hydrogencarbonate and water and then dried overanhydrous sodium sulfate. After the chloroform was removed in vacuo, theresidue was purified by a silica gel column chromatography (silica gel30 g, chloroform:methanol=200:0-4) and furthermore solidified usingdiethyl ether and hexane to afford 1.04 g of the intermediate compound(70).

(NMR Data for the Intermediate Compound (70))

¹H-NMR (CD₃OD) δ ppm: 7.74-7.78 (2H, m), 7.56-7.69 (2H, m), 7.22-7.42(9H, m), 7.10-7.17 (4H, m), 6.80-6.86 (4H, m), 6.11 (1H, s), 5.18-5.31(1H, m), 4.96-5.08 (2H, m), 4.13-4.56 (7H, m), 4.01-4.13 (1H, m),3.88-3.95 (1H, m), 3.75 (6H, s), 2.37-2.74 (6H, m), 1.96-2.17 (3H, m),1.50-1.96 (10H, m), 1.10-1.50 (28H, m), 0.76-0.99 (27H, m)

SYNTHESIS EXAMPLE 116

To a solution of the intermediate compound (70) (0.97 g) obtained inSynthesis Example 115 in DMF (10 ml) was added diethylamine (1 ml) andthe mixture was stirred at room temperature for 1.5 hours. After thesolvent was removed in vacuo, the residue was dissolved in methanol (40ml), 5% palladium carbon (0.10 g) was added and the mixture was stirredunder hydrogen atmosphere for 3 hours. The palladium carbon was filteredoff and the methanol was removed in vacuo, and then the residue waspurified by a silica gel column chromatography (silica gel 18 g,chloroform:methanol=200:0-20) and furthermore solidified with ethylacetate-hexane to afford 0.53 g of the intermediate compound (71).

(NMR Data for the Intermediate Compound (71))

¹H-NMR (CD₃OD) δ ppm: 7.15 (4H, dd, J=4.9, 8.3 Hz), 6.86 (4H, dd, J=3.4,8.8 Hz), 6.09 (1H, s), 5.21-5.40 (1H, m), 4.37-4.49 (2H, m), 4.19 (1H,d, J=7.3 Hz), 4.15-4.26 (1H, m), 4.04 (1H, d, J=5.9 Hz), 3.91-4.00 (1H,m), 3.77 (6H, s), 2.86-3.02 (1H, m), 2.70-2.80 (1H, m), 2.47-2.60 (2H,m), 2.32-2.44 (2H, m), 1.82-2.01 (1H, m), 1.49-1.82 (8H, m), 1.44 (9H,2s), 1.14-1.49 (22H, m), 0.80-1.07 (27H, m)

SYNTHESIS EXAMPLE 117

To a solution of the intermediate compound (71) (0.50 g) obtained inSynthesis Example 116 in THF (95 ml) were added N-methylmorpholine (0.09ml) and HOBt.monohydrate (0.25 g) to form Solution A. To a mixed solventof THF (190 ml) and DMF (95 ml) were added in turn cesium chloride (0.70g), potassium chloride (0.28 g) and WSCI (0.71 g) to form Solution B.Solution A was added dropwise to Solution B at room temperature over 20minutes while stirring and the mixture was further stirred at roomtemperature for 5 days. The reaction solution was diluted with ethylacetate (100 ml) and the mixture was washed in turn with water, 5%aqueous sodium hydrogencarbonate, water, 10% aqueous citric acid andwater and then dried over anhydrous sodium sulfate. After the solventwas removed in vacuo, the residue was purified by a silica gel columnchromatography (silica gel 30 g, chloroform:methanol=200:0-6) to afford0.44 g of the cyclic depsipeptide (25) of the invention.

(NMR Data for the Cyclic Depsipeptide (25))

¹H-NMR (CD₃OD) δ ppm: 7.10-7.17 (4H, m), 6.81-6.89 (4H, m), 6.08 (1H,s), 5.07-5.23 (1H, m), 4.35-4.54 (3H, m), 4.23-4.32 (2H, m), 3.89-3.94(1H, m), 3.78 (3H, s), 3.77 (3H, s), 2.71-3.04 (3H, m), 2.35-2.53 (4H,m), 2.13-2.45 (1H, m), 1.92-2.08 (2H, m), 1.50-1.81 (9H, m), 1.44 (9H,s), 1.21-1.50 (19H, m), 0.83-1.02 (27H, m)

SYNTHESIS EXAMPLE 118

A solution of the cyclic depsipeptide (25) (0.40 g) obtained inSynthesis Example 117 in TFA (5 ml) was stirred at room temperature for2 hours. After the solvent was removed in vacuo, the residue wasneutralized with 5% aqueous sodium hydrogencarbonate and extracted witha 10% methanolic solution of chloroform. The organic layer was driedover anhydrous sodium sulfate and concentrated and then purified by asilica gel column chromatography (silica gel 20 g,chloroform:methanol=100:0-50) and furthermore solidified with diethylether-hexane to afford 0.25 g of the cyclic depsipeptide (26) of theinvention.

(NMR Data for the Cyclic Depsipeptide (26))

¹H-NMR (CD₃OD) δ ppm: 5.08 (1H, s), 4.37-4.64 (5H, m), 3.94-3.98 (1H,m), 2.36-3.16 (3H, m), 2.16-2.36 (4H, m), 1.86-2.06 (3H, m), 1.47-1.80(7H, m), 1.15-1.44 (21H, m), 0.83-1.08 (27H, m)

SYNTHESIS EXAMPLE 119

The desired alcohol (0.86 g) was obtained from(R)-(+)-1,2-epoxy-3-undecyloxypropane (2.71 g) in the same manner asdescribed in Synthesis Example 105.

¹H-NMR (CDCl₃) δ ppm: 7.28-7.41 (5H, m), 5.16 (2H, s), 4.18-4.26 (1H,m), 3.35-3.49 (4H, m), 2.87 (1H, br s), 2.58 (2H, d, J=6.3 Hz), 1.56(2H, qui., J=6.8 Hz), 1.20-1.35 (16H, m), 0.88 (3H, t, J=6.8 Hz)

SYNTHESIS EXAMPLE 120

The desired intermediate compound (1.50 g) was obtained starting fromthe alcohol (0.84 g) obtained in Synthesis Example 119 In the samemanner as described in Synthesis Example 106.

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=7.3 Hz), 7.60 (2H, d, J=5.9 Hz),7.40 (2H, t, J=7.3 Hz), 7.27-7.37 (7H, m), 5.39-5.49 (1H, m), 5.32 (1H,d, J=8.8 Hz), 5.12 (2H, s), 4.36-4.41 (2H, m), 4.32 (1H, dd, J=4.6, 8.8Hz), 4.23 (1H, t, J=6.8 Hz), 3.55 (1H, dd, J=5.1, 11 Hz), 3.49 (1H, dd,J=4.4, 10 Hz), 3.31-3.45 (2H, m), 2.74 (2H, d, J=6.8 Hz), 1.88 (1H, brs), 1.36-1.56 (2H, m), 1.06-1.34 (18H, m), 0.88 (3H, t, J=6.8 Hz),0.85-0.94 (6H, m)

SYNTHESIS EXAMPLE 121

The desired chain depsipeptide (0.77 g) was obtained from theintermediate compound (0.35 g) obtained in Synthesis Example 120 in thesame manner as described in Synthesis Example 107.

However, instead of the tripeptide (6), there was used the hexapeptide(0.64 g) which was obtained by debenzylating the hexapeptide (1)obtained in Synthesis Example 49 in the same manner as described inSynthesis Example 17.

¹H-NMR (CDCl₃) δ ppm: 8.48 (1H, d, J=8.8 Hz), 7.72 (2H, d, J=7.3 Hz),7.64-7.78 (1H, m), 7.51-7.62 (2H, m), 7.41 (1H, d, J=8.3 Hz), 7.36 (2H,t, J=7.3 Hz), 7.20 (2H, d, J=8.8 Hz), 7.16 (2H, d, J=8.3 Hz), 7.13-7.45(8H, m), 7.10 (1H, d, J=8.8 Hz), 7.00 (1H, d, J=8.3 Hz), 6.93 (1H, d,J=5.4 Hz), 6.85 (4H, d, J=8.8 Hz), 6.61 (1H, d, J=7.8 Hz), 6.25 (1H, d,J=8.3 Hz), 5.29-5.41 (1H, m), 4.99-5.17 (3H, m), 4.27-4.64 (5H, m),4.10-4.24 (2H, m), 3.91-4.04 (2H, m), 3.77 (3H, s), 3.76 (3H, s),3.25-3.57 (4H, m), 3.14 (1H, dd, J=3.4, 15 Hz), 2.62-2.78 (3H, m),2.44-2.55 (1H, m), 2.30-2.41 (1H, m), 2.14 (1H, br s), 1.42 (9H, S),1.07-2.08 (32H, m), 0.65-1.05 (33H, m)

SYNTHESIS EXAMPLE 122

The desired cyclic depsipeptide (27) (0.30 g) was obtained from thechain depsipeptide (0.39 g) obtained in Synthesis Example 121 in thesame manner as described in Synthesis Example 52 (3).

(Data for the Cyclic Depsipeptide (27))

¹H-NMR (CDCl₃) δ ppm: 7.71 (1H, d, J=8.8 Hz), 7.36 (1H, d, J=8.3 Hz),7.17 (1H, d, J=8.8 Hz), 7.14 (2H, d, J=8.8 Hz), 7.07-7.21 (2H, m),6.98-7.07 (2H, m), 6.91 (1H, d, J=8.3 Hz), 6.85 (4H, dd, J=1.5, 8.3 Hz),6.70 (1H, br s), 6.21 (1H, d, J=8.3 Hz), 5.07-5.17 (2H, m), 4.34-4.49(3H, m), 4.20 (1H, dd, J=4.4, 7.8 Hz), 4.08-4.16 (1H, m), 3.87 (1H, t,J=5.4 Hz), 3.79 (6H, 2s), 3.75-3.84 (2H, m), 3.53 (1H, dd, J=3.9, 10Hz), 3.36-3.51 (2H, m), 3.14 (1H, dd, J=4.9, 16 Hz), 2.68 (1H, dd,J=5.4, 15 Hz), 2.38-2.53 (2H, m), 2.16-2.37 (2H, m), 1.47-2.15 (16H, m),1.40 (9H, s), 1.09-1.46 (16H, m), 0.78-1.04 (33H, m)

SYNTHESIS EXAMPLE 123

The desired cyclic depsipeptide (28) (0.16 g) was obtained from thecyclic despipeptide (27) (0.28 g) obtained in Synthesis Example 122 inthe same manner as described in Synthesis Example 12.

(Data for the Cyclic Depsipeptide (28))

¹H-NMR (DMSO-d₆+TFA) δ ppm: 8.40 (1H, d, J=8.3 Hz), 8.31 (1H, d, J=6.3Hz), 8.16 (1H, d, J=7.3 Hz), 7.99 (1H, d, J=5.9 Hz), 7.96 (1H, d, J=7.3Hz), 7.84 (1H, d, J=8.3 Hz), 7.79 (1H, d, J=8.3 Hz), 7.31 (1H, s), 6.85(1H, s), 5.07-5.16 (1H, m), 4.44-4.59 (2H, m), 4.12-4.29 (4H, m), 4.07(1H, t, J=7.6 Hz), 3.48-3.55 (1H, m), 3.26-3.46 (3H, m), 2.58-2.72 (2H,m), 2.35-2.46 (2H, m), 1.95-2.16 (3H, m), 1.68-1.94 (3H, m), 1.32-1.65(11H, m), 1.11-1.31 (18H, m), 0.71-0.96 (33H, m)

SYNTHESIS EXAMPLE 124

The desired alcohol (0.47 g) was obtained from (R)-1,2-epoxyoctadecane(3.00 g) in the same manner as described in Synthesis Example 105.

¹H-NMR (CDCl₃) δ ppm: 7.29-7.42 (5H, m), 5.16 (2H, s), 3.97-4.07 (1H,m), 2.84 (1H, br s), 2.56 (1H, dd, J=3.2, 16 Hz), 2.46 (1H, dd, J=9.0,17 Hz), 1.37-1.67 (4H, m), 1.06-1.36 (26H, m), 0.88 (3H, t, J=6.8 Hz)

SYNTHESIS EXAMPLE 125

The desired intermediate compound (0.82 g) was obtained from the alcohol(0.45 g) obtained in Synthesis Example 124 in the same manner asdescribed in Synthesis Example 106.

(Data for the Above Intermediate Compound)

¹H-NMR (CDCl₃) δ ppm: 7.76 (2H, d, J=7.8 Hz), 7.59 (2H, d, J=5.9 Hz),7.39 (2H, t, J=7.6 Hz), 7.26-7.36 (7H, m), 5.22-5.36 (2H, m), 5.10 (2H,s), 4.34-4.42 (2H, m), 4.31 (1H, dd, J=4.4, 8.8 Hz), 4.22 (1H, t, J=7.1Hz), 2.69 (1H, dd, J=6.8, 16 Hz), 2.59 (1H, dd, J=5.6, 15 Hz), 1.89 (1H,br s), 1.61 (2H, br s), 1.05-1.48 (30H, m), 0.85-1.00 (6H, m), 0.88 (3H,t, J=6.6 Hz)

SYNTHESIS EXAMPLE 126

The desired chain depsipeptide (0.69 g) was obtained from theintermediate compound (0.40 g) obtained in Synthesis Example 125 in thesame manner as described in Synthesis Example 121.

¹H-NMR (CDCl₃) δ ppm: 8.48 (1H, d, J=8.8 Hz), 7.73 (2H, d, J=7.3 Hz),7.69 (1H, d, J=8.8 Hz), 7.55 (2H, dd, J=3.2, 6.8 Hz), 7.42 (1H, d, J=8.3Hz), 7.37 (2H, t, J=7.3 Hz), 7.20 (2H, d, J=8.3 Hz), 7.17 (2H, d, J=8.3Hz), 7.15-7.45 (8H, m), 7.04-7.13 (1H, m), 7.01 (1H, d, J=8.3 Hz), 6.93(1H, d, J=5.9 Hz), 6.85 (4H, d, J=8.3 Hz), 6.54 (1H, d, J=7.8 Hz), 6.25(1H, d, J=8.3 Hz), 4.98-5.23 (4H, m), 4.27-4.69 (5H, m), 4.10-4.24 (2H,m), 3.92-4.04 (2H, m), 3.77 (3H, s), 3.76 (3H, s), 3.13 (1H, dd, J=3.4,15 Hz), 2.62-2.76 (2H, m), 2.43-2.58 (2H, m), 2.31-2.41 (1H, m), 2.14(1H, br s), 1.68-2.08 (5H, m), 1.42 (9H, S), 1.05-1.67 (39H, m),0.75-1.04 (33H, m)

SYNTHESIS EXAMPLE 127

The desired cyclic depsipeptide (27) (0.28 g) was obtained from thechain depsipeptide (0.32 g) obtained in Synthesis Example 126 in thesame manner as described in Synthesis Example 122.

(Data for the Cyclic Depsipeptide (27))

¹H-NMR (CDCl₃) δ ppm: 7.64 (1H, d, J=8.3 Hz), 7.31 (1H, d, J=7.3 Hz),7.17 (1H, d, J=8.8 Hz), 7.14 (2H, d, J=8.8 Hz), 7.07-7.19 (2H, m), 7.01(1H, d, J=9.3 Hz), 6.96 (1H, d, J=8.8 Hz), 6.90 (1H, d, J=7.3 Hz), 6.85(4H, d, J=7.8 Hz), 6.78 (1H, br s), 6.20 (1H, d, J=8.3 Hz), 5.05-5.15(1H, m), 4.95-5.04 (1H, m), 4.29-4.49 (3H, m), 4.18 (1H, dd, J=4.4, 12Hz), 4.09-4.21 (1H, m), 3.87 (1H, t, J=5.4 Hz), 3.79 (6H, 2s), 3.15 (1H,dd, J=4.4, 17 Hz), 2.63 (1H, dd, J=4.4, 16 Hz), 2.51 (1H, dd, J=9.8, 17Hz), 2.30-2.40 (2H, m), 2.19-2.28 (1H, m), 1.54-2.13 (15H, m), 1.40 (9H,s), 1.04-1.48 (30H, m), 0.77-1.02 (33H, m)

SYNTHESIS EXAMPLE 128

The desired cyclic depsipeptide (28) (0.14 g) was obtained from thecyclic depsipeptide (27) (0.27 g) obtained in Synthesis Example 127 inthe same manner as described in Synthesis Example 123.

(Data for the Cyclic Depsipeptide (28))

¹H-NMR (DMSO-d₆) δ ppm: 12.30 (1H, br s), 8.38 (1H, d, J=6.8 Hz), 8.30(1H, d, J=7.8 Hz), 8.11 (1H, d, J=7.3 Hz), 7.86 (1H, d, J=8.8 Hz),7.81-7.96 (3H, m), 7.30 (1H, s), 6.86 (1H, s), 4.91-5.00 (1H, m),4.45-4.61 (2H, m), 4.12-4.27 (4H, m), 4.08 (1H, t, J=7.3 Hz), 2.65 (2H,d, J=6.3 Hz), 2.27-2.44 (2H, m), 1.96-2.14 (3H, m), 1.67-1.93 (3H, m),1.08-1.64 (41H, m), 0.68-0.92 (33H, m)

EXAMPLE 1

It will be explained below that the cyclic depsipeptides of theinvention show a promoting activity on the production of apolipoproteinE in Hep G2 cells, together with the test procedures used.

The compounds used in the following Examples are as defined below:

-   -   R=isopropyl, n=7 Compound 1    -   R=isopropyl, n=8 Compound 2    -   R=isopropyl, n=9 Compound 3

The above Compounds 1, 2 and 3 were obtained according to the processfor the production of the Compounds 3, 1 and 4 disclosed in WO 95/32990,respectively; that is to say, by culturing Bacillus sp. No. 4691 strain(FERM BP-5101) capable of producing the Compounds 1-3 of the presentinvention and extracting and purifying the said cyclic depsipeptidesfrom the cultured broth.

First, 1 ml portions of Hep G2 cells at 1×10⁵ cells/ml suspended inDulbecco's modified Eagle medium (manufactured by Nissui Seiyaku Co.,Ltd.; hereinafter referred to as “D-MEM medium”) containing 10% fetalbovine serum were poured into a 24-well tissue culture plate andcultivation was carried out at 37° C. under atmosphere of a mixed gascomposed of 5% carbon dioxide and 95% air. After 3 days, the medium wasremoved by means of a pipette, 1 ml of fresh D-MEM medium was added andfurther 10 μl of a methanolic solution of the cyclic depsipeptide of theinvention, or Compound 1, 2 or 3, at the concentration as shown in Table2 was added. After 18 hours, the medium was again replaced (D-MEMmedium), 10 μl of a methanolic solution of the cyclic depsipeptide wasadded and then cultivation was continued at 37° C. for 8 hours to obtainthe cultured broths 1-6. The apolipoprotein E produced in the culturedbroths 1-6 was assayed by means of an enzyme immunoassay method.

The composition of the buffers used in the enzyme immunoassay issummarized in the following Table 1, wherein PBS representsphosphate-buffered saline, PBS-T represents phosphate-buffered salinehaving incorporated Tween 20 and the blocking solution is the phosphatebuffer containing the immunosuppressive agent derived from lactoprotein“Block Ace”, manufactured by Dainippon Pharmaceutical Co., Ltd.

TABLE 1 PBS (pH 7.2) KH₂PO₄ 0.2 g Na₂HPO₄.12H₂O 2.9 g NaCl 8.0 g KCl 0.2g Distilled water q.s. Total amount 1000 ml PBS-T (pH 7.2) KH₂PO₄ 0.2 gNa₂HPO₄.12H₂O 2.9 g NaCl 8.0 g KCl 0.2 g Tween 20 0.5 g Distilled waterq.s. Total amount 1000 ml Blocking soln. (pH 7.2) Block Ace 250 mlKH₂PO₄ 0.2 g Na₂HPO₄.12H₂O 2.9 g NaCl 8.0 g KCl 0.2 g Distilled waterq.s. Total amount 1000 ml

The mouse antihuman apolipoprotein E monocional antibody (manufacturedby BYOSIS, S. A., France) was dissolved in a 0.05M aqueous sodiumhydrogencarbonate solution (pH 9.5) at a concentration of 5 μl/ml. 50 μlof this solution was poured in portions into a Nunk immunoplate, whichwas then allowed to stand at 4° C. for 16 hours. After washing thricewith 300 μl of PBS, 300 μl of the blocking solution was added and themixture was allowed to stand at 37° C. for 2 hours.

It was again washed three times with 300 μl of PBS, 50 μl of the abovecultured broth 1 was added and the mixture was allowed to stand at roomtemperature for 2 hours. After washing three times with 300 μl of PBS-T,50 μl of a 3000-fold diluted solution (10% aqueous Block Ace solution)of goat anti-apolipoprotein E polyclonal antibody (manufactured byChemicon Co., Ltd., U.S.A.) was added and the mixture was allowed tostand at room temperature for 2 hours. The mixture was washed threetimes with 300 μl of PBS-T, a 5000-fold diluted solution (a 10% aqueoussolution of Block Ace) of a peroxidase-labeled anti-goat IgG polyclonalantibody (manufactured by Bindingsite Co., Ltd., U.K.) was added and themixture was allowed to stand at room temperature for 2 hours. Afterwashing five times with 300 μl of PBS-T, 100 μl of a coloring solution(Composition: 0.1M potassium citrate (pH 4.5) 1 ml, 30% aqueous hydrogenperoxide 0.4 μl, orthophenylenediamine 1 mg) was added and the mixturewas allowed to stand as such for 2 minutes. The reaction wasdiscontinued by the addition of 100 μl of 2N sulfuric acid, andabsorbance at 490 nm was measured using absorbance at 650 nm as acontrol. An absolute amount of apolipoprotein E in cultured broths 1-6was determined upon a calibration curve drawn up when a commerciallyavailable apolipoprotein E (Chemicon Co., Ltd., U.S.A.) was used as astandard.

The same procedure as described in Example 1 was carried out except thatmethanol was added instead of the methanolic solution of the cyclicdepsipeptide to measure an apolipoprotein E amount, which was used as acontrol.

A relative apolipoprotein E amount of each sample was represented interms of a relative value (t) when the control was defined as 100.

As shown in Table 2, it was proved that Compounds 1-3 of the inventionhave a potent activity of promoting the production of apolipoprotein Eat 1 or 5 μM.

TABLE 2 Relative amount of Compound Conc. (μM) apolipoprotein E (%) 1 1207 5 256 2 1 110 5 243 3 1 147 5 278 Control 0 100

As is seen from the above results, the cyclic depsipeptide of theinvention highly promotes the production of apolipoprotein E in Hep G2cells and thus it is useful as a novel type of a therapeutic agent forneurologic damages or an antidementia agent.

EXAMPLE 2

It will be explained below that the cyclic depsipeptides of theinvention represented by the above formula (1′) show a promotingactivity on the production of apolipoprotein E, an inhibitory activityon the production of apolipoprotein B and a promoting activity on theproduction of apolipoprotein A1 in Hep G2 cells, together with the testprocedures used.

First, 1 ml portions of Hep G2 cells at 1×10⁵ cells/ml suspended inD-MEM medium containing 10% fetal bovine serum were poured into a24-well tissue culture plate and cultivation was carried out at 37° C.under atmosphere of a mixed gas composed of 5% carbon dioxide and 95%air for 3 days. Thereafter, the medium was removed by means of apipette, 1 ml of fresh D-MEM medium was added and cultivation was againcarried out at 37° C. under atmosphere of a mixed gas composed of 5%carbon dioxide and 95% air for one day. Then, the medium was removed bymeans of a pipette and washed three times with 0.5 ml of fresh D-MEMmedium. 1 ml of fresh D-MEM medium was added and further 10 μl of asolution of the cyclic depsipeptide of the invention or Compound 1, 4,5, 6, 7 or 8 dissolved in methanol at the concentration as shown inTable 3 was added. Cultivation was further continued at 37° C. for 7hours to obtain Cultured Broths 7-10. The apolipoprotein E,apolipoprotein B and apolipoprotein Al produced in Cultured Broths 7-10were assayed. Assay for apolipoprotein E was carried out by using thesame procedure as described in Example 1. Assay for apolipoprotein B andapolipoprotein A1 will be as described below.

1) Assay for Apolipoprotein B

The sheep antihuman apolipoprotein B IgG fraction (manufactured byBindingsite Co., Ltd., U.K.) was dissolved in a 0.05M aqueous sodiumhydrogencarbonate solution (pH 9.5) at a concentration of 10 μg/ml. 50μl of this solution was poured in portions into a Nunk immunoplate,which was then allowed to stand at 4° C. for 16 hours. After washingthree times with 300 μl of PBS, 300 μl of the blocking solution wasadded and the mixture was allowed to stand at 37° C. for 2 hours. It wasagain washed three times with 300 μl of PBS, 50 μl of the culturesolution (the above Cultured Broths 7-10 diluted with 10% Block Ace to3.3 times volumes, respectively) was added and the mixture was allowedto stand at room temperature for 2 hours. After washing three times with300 μl of PBS-T, 50 μl of a 0.5% solution of sheep antihumanapolipoprotein B peroxidase labeled preparation (manufactured byBindingsite Co., Ltd., U.K.) (10% aqueous “Block Ace” solution) wasadded and the mixture was allowed to stand at room temperature for 2hours. After washing five times with 300 μl of PBS-T, 100 μl of acoloring solution (Composition: 0.1M potassium citrate (pH 4.5) 1 ml,30% aqueous hydrogen peroxide 0.4 μl, orthophenylene-diamine 1 mg) wasadded and the mixture was allowed to stand as such for 2 minutes. Thereaction was discontinued by the addition of 100 μl of 2N sulfuric acid,and absorbance at 490 nm was measured using absorbance at 650 nm as acontrol. The absorbance obtained was defined as that of apolipoproteinB. An absolute amount of apolipoprotein B was determined upon acalibration curve drawn up when a commercially available low densitylipoprotein (manufactured by Sigma, U.S.A.) was used as a standard.

The same procedure as described in Example 1 was carried out except thatmethanol was added instead of the methanolic solution of the cyclicdepsipeptide to measure an apolipoprotein B amount, which was used as acontrol.

A relative apolipoprotein B amount of the present cyclic depsipeptideswas represented in terms of a relative value (%) when the control wasdefined as 100.

2) Assay for Apolipoprotein A1

The mouse antihuman apolipoprotein A1 monoclonal antibody (manufacturedby Medix Biotec, U.S.A.) was dissolved in a 0.05M aqueous sodiumhydrogencarbonate solution (pH 9.5) at a concentration of 10 μg/ml. 50μl of this solution was poured in portions into a Nunk immunoplate,which was then allowed to stand at 4° C. for 16 hours. After washingthree times with 300 μl of PBS, 300 μl of the blocking solution wasadded and the mixture was allowed to stand at 37° C. for 2 hours andthen at 4° C. for 16 hours.

It was again washed three times with 300 μl of PBS, 100 μl of the aboveCultured Broths 7-10 was added and the mixture was allowed to stand atroom temperature for 2 hours. After washing three times with 300 μl ofPBS-T, 50 μl of a 2000-fold diluted solution of sheepanti-apolipoprotein A1 peroxidase labeled preparation (manufactured byBindingsite Co., Ltd., U.K.) (10% aqueous “Block Ace” solution) wasadded and the mixture was allowed to stand at room temperature for 1.5hours. After washing three times with 300 μl of PBS-T, 100 μl of acoloring solution (Composition: 0.1M potassium citrate (pH 4.5) 1 ml,30% aqueous hydrogen peroxide 0.4 μl, orthophenylenediamine 1 mg) wasadded and the mixture was allowed to stand as such for 2 minutes. Thereaction was discontinued by the addition of 100 μl of 2N sulfuric acid,and absorbance at 490 nm was measured using absorbance at 650 nm as acontrol. An absolute amount of apolipoprotein A1 was determined upon acalibration curve drawn up when a commercially available apolipoproteinAl (manufactured by Sigma, U.S.A.) was used as a standard.

The same procedure as described in Example 2 was carried out except thatmethanol was added instead of the methanolic solution of the cyclicdepsipeptide to measure an apolipoprotein Al amount, which was used as acontrol.

A relative apolipoprotein Al amount of the present cyclic depsipeptideswas represented in terms of a relative value (%) when the control wasdefined as 100.

As shown in Table 3, it was proved that the cyclic depsipeptides of theinvention have a potent activity of promoting the production ofapolipoprotein E at 1 or 5 μM. Also, it was proved that Compounds 3-7highly promote the production of apolipoprotein A1 and also have apotent inhibitory activity on the production of apolipoprotein B.

TABLE 3 Relative Relative Relative amount of amount of amount ofapolipo- apolipo- apolipo- Conc. protein E protein B protein A1 (μM) (%)(%) (%) Compound 4 1 457 43 147 Compound 4 5 1042  25 161 Compound 3 1359 64 124 Compound 3 5 851 21 146 Compound 5 1 132 91 123 Compound 6 1198 72 142 Compound 7 1 163 76 135 Compound 8 1 423 71  96 Control 0 100100  100

As is seen from the above results, the cyclic depsipeptide of theinvention represented by the above formula (1) markedly promotes theproduction of apolipoprotein E or apolipoprotein A1 in Hep G2 cells at alow concentration and markedly inhibits the production of apolipoproteinB, and thus it is useful as a therapeutic agent for hyperlipemia.

PREPARATION EXAMPLES Preparation Example 1 Tablets (per Tablet)

Compound 4 20 mg Magnesium silicate 20 mg Lactose 98.5 mgHydroxypropylcellulose 7.5 mg Magnesium stearate 1 mg Hydrogenatedvegetable oil 3 mg Total 150 mg

Compound 4, magnesium silicate and lactose were admixed and kneaded withan alcoholic solution of hydroxypropylcellulose and then granulated toappropriate particle size, dried, and sized. Then, magnesium stearateand hydrogenated vegetable oil were added and blended to form uniformgranules. The granules were then prepared to tablets, each having adiameter of 7.0 mm, a weight of 150 mg and a hardness of 6 kg, by meansof a rotary tableting machine.

Preparation Example 2 Granules

Compound 4 10 mg Magnesium oxide 40 mg Calcium hydrogenphosphate 38 mgLactose 10 mg Hydroxypropylcellulose 20 mg

All the materials except for hydroxypropyl-cellulose were uniformlyadmixed, kneaded with an alcoholic solution of hydroxypropylcelluloseand then granulated by means of an extrusion granulation machine anddried to form granules. The granules were sized so as to pass through a12 mesh sieve and remain on a 48 mesh sieve, thereby forming granules.

Preparation Example 3 Syrups

Compound 4 1.000 g Sucrose 30.000 g D-Sorbitol 70 w/v % 25.000 g Ethylparaoxybenzoate 0.030 g Propyl paraoxybenzoate 0.015 g Flavoring agent0.200 g Glycerol 0.150 g 96% Ethanol 0.500 g Purified water q.s. Total100 ml

Sucrose, D-sorbitol, ethyl paraoxybenzoate, propyl paraoxybenzoate andCompound 4 were dissolved in 60 g of purified water (warm water). Aftercooling, a solution of flavoring agent in glycerol and ethanol was addedand then to the mixture was added purified water to make up a volume to100 ml.

Preparation Example 4 Injections

Sodium salt of Compound 4 10.0 mg Sodium chloride 81.0 mg Sodiumhydrogencarbonate 8.40 mg Distilled water for injection q.s. Total 10.0ml

Sodium hydrogencarbonate, sodium chloride and sodium salt of Compound 4were dissolved in distilled water to make up a total amount to 10.0 ml.

Preparation Example 5 Suppositories

Compound 4 2 g Macrogol 4000 20 g Glycerol 78 g Total 100 g

Compound 4 was dissolved in glycerol and then macrogol 4000 was addedand dissolved by warming. Then, the mixture was injected into asuppository die and solidified by cooling to prepare suppositories, eachweighing 1.5 g.

Industrial Applicability

The cyclic depsipeptides of the present invention have a promotingactivity on the production of apolipoprotein E, an inhibitory activityon the production of apolipoprotein B and a promoting activity on theproduction of apolipoprotein A1. Since apolipoprotein E has a repairingaction on neurologic damages, the cyclic depsipeptides of the inventionhaving an activity of promoting the production of apolipoprotein E areuseful as a therapeutic agent for neurologic damages, especially anantidementia agent. Moreover, since apolipoprotein B is a mainapolipoprotein of a low density lipoprotein cholesterol (LDLcholesterol) known as a “bad” cholesterol and apolipoprotein A1 is amain apolipoprotein of a high density lipoprotein cholesterol (HDLcholesterol) known as a “good” cholesterol, the cyclic depsipeptides ofthe invention having an action of inhibiting the production ofapolipoprotein B and an action of promoting the production ofapolipoprotein A1 are useful as a therapeutic agent for hyperlipemia.

1. A method for promoting production of apolipoprotein E, whichcomprises administering to a patient in need thereof one or more cyclicdepsipeptide of the formula (1) as an active ingredient:

wherein: R is a straight or branched alkyl group of 5 to 20 carbonatoms, or a straight or branched alkoxymethyl group of 5-15 carbonatoms; A, B, D, E and F are each independently a residue of an aminoacid selected from the group consisting of alanine, valine, leucine,isoleucine, serine, threonine, lysine, hydroxylysine, arginine,cysteine, methionine, phenylalanine, tyrosine, tryptophan, histidine,proline, 4-hydroxyproline, piperizine-4-carboxylic acid, homoproline,octahydroindole-2-carboxylic acid, norvaline, norleucine,α-t-butylglycine, cyclohexylglycine, azetidine-2-carboxylic acid,3-(3-pyridyl)alanine, (3-N-methyl)piperizylalanine,3-(2-naphthyl)alanine,βcyclohexylalanine, β-t-butylalanine,9-anthracenylalanine, α-methylalanine, 2-aminobutanoic acid, and anN—(C₁-C₄) alkyl derivative of any of the above foregoing amino acidresidues; W and Z are independently a residue of an amino acid selectedfrom the group consisting of aspartic acid, asparagine, glutamic acidand glutamine; and m and n are independently 0 or 1, wherein any freeamino group, any free carboxyl group or any free ω-carbamido group ofsaid amino acid residue in A, B, D, E, F, W and Z is optionallyprotected by a protecting group used in peptide chemistry. furtherwherein when said amino acid residue in A, B, D, E, F, W and Z is aresidue of lysine, hydroxylysine, glutamic acid or aspartic acid, theamino group or carboxyl group capable of being bound to an adjacentamino acid by peptide linkage is optionally located at either theα-position or the ω-position, or a pharmacologically acceptable saltthereof.
 2. The method as claimed in claim 1, which comprisesadministering as an active ingredient one or more cyclic depsipeptidesof the formula (1) wherein A is isoleucine or alanine; B is leucine,alanine, β-t-butylalanine, valine or phenylalanine; D is valine oralanine; E is leucine, alanine, β-t-butylalanine, valine orphenylalanine; F is leucine, alanine, β-t-butylalanine, valine orphenylalanine; W is aspartic acid or glutamic acid; and Z is glutamineor asparagine; m and n are 0 or 1; and R is a straight alkyl group oralkoxymethyl group of 6 to 12 carbon atoms, or a pharmaceuticallyacceptable salt thereof.
 3. The method as claimed in claim 1, whichcomprises administering as an active ingredient one or more cyclicdepsipeptides of the formula (1) wherein A is isoleucine or alanine; Bis D-leucine, D-alanine, D-β-t-butylalanine, D valine orD-phenylalanine; E is D-leucine, D-alanine, D-β-t butylalanine, D-valineor D-phenylalanine; F is leucine, alanine, β-butylalanine, valine orphenylalanine; W is aspartic acid or glutamic acid; and Z is glutamineor asparagine; m and n are 0 or 1; and R is a straight alkyl group oralkoxymethyl group of 6 to 12 carbon atoms, or a pharmaceuticallyacceptable salt thereof.
 4. The method as claimed in claim 1, whereinsaid one or more cyclic depsipeptides of the formula (1) have any of thefollowing formulae:

wherein R is as defined above.
 5. The method as claimed in claim 1,wherein said one or more cyclic depsipeptides of the formula (1) havethe formula:

wherein, R₂ is methyl or isopropyl; R₃ is a direct bond or —OCH₂—; and qis an integer of 2-10.
 6. A cyclic depsipeptide of the formula (1′):

wherein: R is a straight or branched alkyl group of 5-20 carbon atoms,or a straight or branched alkoxymethyl group of 5-15 carbon atoms; A, B,D, E and F are each independently a residue of an amino acid selectedfrom the group consisting of alanine, valine, leucine, isoleucine,serine, threonine, lysine, hydroxylysine, arginine, cysteine,methionine, phenylalanine, tyrosine, tryptophan, histidine, proline,4-hydroxyproline, piperizine-4-carboxylic acid, homoproline,octahydroindole-2-carboxylic acid, norvaline, norleucine,α-t-butylglycine, cyclohexylglycine, azetidine-2-carboxylic acid,3-(3-pyridyl)alanine, (3-N-methyl)piperizylalanine,3-(2-naphthyl)alanine,β-cyclohexylalanine, β-t-butylalanine,9-anthracenylalanine, α-methylalanine, 2-aminobutanoic acid, and anN—(C₁-C₄) alkyl derivative of any of the foregoing amino acid residues;W and Z are independently a residue of an amino acid selected from thegroup consisting of aspartic acid, asparagine, glutamic acid andglutamine; and m and n are independently 0 or 1, wherein any free aminogroup, any free carboxyl group or any free ω-carbamido group of saidamino acid residue in A, B, D, E, F, W, and Z is optionally protected bya protecting group used in peptide chemistry, further wherein when saidamino acid residue in A, B, D, E, F, W and Z is a residue of lysine,hydroxylysine, glutamic acid or aspartic acid, the amino group orcarboxyl group capable of being bound to an adjacent amino acid bypeptide linkage is optionally located at either the α-position or theω-position, provided that there are excluded the cases wherein m and nare 1, A is isoleucine, B is leucine, W is aspartic acid, D is valine, Eis leucine, F is leucine, Z is glutamic acid or glutamine and R′ is agroup of the formula R₁-(CH₂)_(p)— wherein R₁ is methyl, isopropyl,sec-butyl or isobutyl and p is an integer of 5-15, or apharmacologically acceptable salt thereof.
 7. The cyclic depsipeptide asclaimed in claim 6, wherein A is isoleucine or alanine, B is leucine,alanine, β-t-butylalanine, valine or phenylalanine, D is valine oralanine, E is leucine, alanine, β-t-butylalanine, valine orphenylalanine, F is leucine, alanine, β-t-butylalanine, valine orphenylalanine, W is aspartic acid or glutamic acid, and Z is glutamineor asparagine, m and n are 1, and R′ is a straight alkyl group oralkoxymethyl group of 6-12 carbon atoms, or a pharmaceuticallyacceptable salt thereof.
 8. The cyclic depsipeptide as claimed in claim6, wherein A is isoleucine or alanine, B is D-leucine, D alanine,D-β-t-butylalanine, D-valine or D-phenylalanine, E is D-leucine,D-alanine, D-β-t-butylalanine, D-valine or D phenylalanine, F isleucine, alanine, β-t-butylalanine, valine or phenylalanine, W isasparatic acid or glutamic acid, and Z is glutamine or asparagine, m andn are 1, and R′ is a straight alkyl group or alkoxymethyl group of 6-12carbon atoms, or a pharmaceutically acceptable salt thereof.
 9. Thecyclic depsipeptide as claimed in claim 6 having any of the followingformulae:


10. The cyclic depsipeptide as claimed in claim 6 having the formula:

wherein, R₂ is methyl or isopropyl; R₃ is a direct bond or —OCH₂—; and qis an integer of 2-10.
 11. A pharmaceutical composition which comprisesa cyclic depsipeptide of the formula (1′)

wherein: R′ is a straight of branched alkyl group of 5 to 20 carbonatoms, or a straight or branched alkoxymethyl group of 5 to 15 carbonatoms; A, B, D, E and F are each independently a residue of an aminoacid selected from the group consisting of alanine, valine, leucine,isoleucine, serine, threonine, lysine, hydroxylysine, arginine,cysteine, methionine, phenylalanine, tyrosine, tryptophan, histidine,proline, 4-hydroxyproline, piperizine-4-carboxylic acid, homoproline,octahydroindole-2-carboxylic acid, norvaline, norleucine,β-t-butylglycine, cyclohexylglycine, azetidine-2-carboxylic acid,3-(3-pyridyl)alanine, (3-N-methyl)piperizylalanine,3-(2-naphthyl)alanine, β-cyclohexylalanine, β-t-butylalanine,9-anthracenylalanine, β-methylalanine and 2-aminobutanoic acid or anN—(C₁-C₄)alkyl derivative of said amino acid residue; W and Z areindependently a residue of an amino acid selected from the groupconsisting of aspartic acid, asparagine, glutamic acid and glutamine;and m and n are independently 0 or 1; and wherein a free amino group, afree carboxyl group or a free ω-carbamido group of said amino acidresidue may be protected by a protecting group commonly used in peptidechemistry and, when said amino acid residue in the above A, B, D, E, F,W and Z is a residue of lysine, hydroxylysine, glutamic acid or asparticacid, the amino group or carboxyl group capable of being bound to anadjacent amino acid by peptide linkage may be located at either theα-position or the ω-position; provided that there are excluded the caseswherein m and n are 1, A is isoleucine, B is leucine, W is asparticacid, D is valine, E is leucine, F is leucine, Z is glutamic acid orglutamine and R′ is a group of the formula R₁-CH₂)_(p)— wherein R₁ ismethyl, isopropyl, sec-butyl or isobutyl and p is an integer of 5-15, ora pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable excipient.
 12. A method for inhibiting production ofapolipoprotein B, which comprises administering to a patient in needthereof one or more cyclic depsipeptides as claimed in claim 6 or apharmaceutically acceptable salt thereof.
 13. A method for promotingproduction of apolipoprotein A1, which comprises administering to apatient in need thereof one or more of the cyclic depsipeptides asclaimed in claim 6 or a pharmaceutically acceptable salt thereof.
 14. Amethod for treating hyperlipemia, which comprises administering to apatient in need thereof one or more of the cyclic depsipeptides asclaimed in claim 6 or a pharmaceutically acceptable salt thereof.